WO2002099109A1 - Novel polypeptide, dna encoding the polypeptide and use thereof - Google Patents

Abstract

A novel microorganism Streptomyces sp. which produces pravastatin (a drug for hyperlipidemia) by hydroxylating compactin at the 6β-position; a novel polypeptide produced by the above microorganism; a DNA encoding this polypeptide; a recombinant DNA obtained by integrating the above DNA; a microorganism having the above recombinant DNA transferred thereinto; and a process for producing pravastatin using the above microorganism.

Description

 Description Novel polypeptides, DNAs encoding the polypeptides and their uses

 The present invention relates to a novel polypeptide having 6β-position hydroxylating activity of compactin, a DNA encoding the polypeptide, and uses thereof.

 More specifically, a novel polypeptide that produces brapastatin known as an antihyperlipidemic agent by hydroxylating the ββ position of compactin, a DNA encoding the polypeptide, and a DNA incorporating the DNA The present invention relates to a recombinant DNA, a microorganism into which the recombinant DNA has been introduced, and a method for producing pravastatin using the microorganism. Background art

 In recent years, ischemic heart disease caused by coronary artery stiffening is increasing with the aging of society and the westernization of diet. The incidence of ischemic heart disease is known to increase when serum cholesterol levels exceed a certain level (W. B. Kannel, Ann. Inntern. Med., ΊΑ, 1 (1971)).

 There are cholesterol in the body that is absorbed from the diet and biosynthesized in the body.In humans, the amount of biosynthesis is reported to be three to four times higher than the amount absorbed from the diet. (JMDietschy, N. Engl. J. Med., 2S2, 1179 (1970)). Therefore, if cholesterol biosynthesis is suppressed, it is expected that serum cholesterol levels will be reduced and that preventive and therapeutic effects for ischemic heart disease will be obtained.

Cholesterol is biosynthesized in vivo through a complex reaction of 20 or more steps starting from acetyl CoA.

The HMG-CoA reductase, which reduces 3-hydroxymethyl 3-A-HM-CoA, is the rate-limiting enzyme. As a substance inhibiting this HMG-CoA reductase, compactin produced by Penicillium citrmum, a kind of blue mold, is known. Compactin has the following formula

で示されるが、 前記 HMG— C o Aに拮抗し、 HM G— C o A還元酵素を特 異的に強力に阻害する。

Which antagonizes HMG-CoA and specifically and strongly inhibits HMG-CoA reductase.

 Even after the discovery of compactin, highly active compactin derivatives were searched for, and active metabolites in urine of dogs to which compactin was administered were identified.

Lum):

が発見された （特公昭 61-13699号、 米国特許第 4346227号、 同第 4410629号 および同第 4448979号） 。 プラバスタチンは優れたコレステロールの生合成 阻害活性および臓器選択的阻害活性を示すことから、 動脈硬化等の虚血性心 疾患を治療または予防する抗高脂血症剤として用いられている。

(JP-B-61-13699, U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,448,799). Pravastatin is used as an antihyperlipidemic agent for treating or preventing ischemic heart disease such as arteriosclerosis because it has excellent cholesterol biosynthesis inhibitory activity and organ-selective inhibitory activity.

 As a method for synthesizing pravastatin, there is known a microbiological method in which compactin is used as a raw material and hydroxylated at position 60 to convert it to pravastatin.

 Japanese Patent Publication Nos. 62-54476 (U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,489,897) include the genus Absidid, the genus Cunninghamellci, the genus Syncepha stmm, the genus Streptomyces. It is described that a specific microorganism belonging to the genus (Streptomyces) has the ability to convert to pravastatin.

 Further, in the specification of Patent No. 2603677 (US Pat. No. 5,179,913) and the specification of Patent No. 2,672,551, Streptomyces' force porphyrus treptomyces carhophilus) SANK 62585 strain (FERM BP-1145) contains 6β-position water of compactin. It describes an enzyme that has oxidase activity and is derived from the microorganism and its gene.

However, these microbiological methods were not sufficient in terms of pravastatin production capacity and production efficiency. Disclosure of the invention

 An object of the present invention is to provide a polypeptide having an enzymatic activity to hydroxylate the 6] -position of compactin and a gene encoding the polypeptide in order to produce pravastatin used as a medicine at low cost. Another object of the present invention is to provide a method for producing pravastatin using a microorganism having the gene incorporated therein. In view of the above problems, the present inventors searched for microorganisms that hydroxylate the 6] -position of compactin, and determined that the specific microorganism isolated from soil added the 6] -position of compactin added to the medium. It has been found that it excels in its ability to produce pravastatin by hydroxylation. The present inventors identified a gene involved in the 6th hydroxylation of compactin from the microorganism, succeeded in closing the gene, and completed the present invention.

 That is, the present invention has the following configuration.

 1. A DNA containing the nucleotide sequence from position 544 to position 1758 in the nucleotide sequence of SEQ ID NO: 1 or a DNA that hybridizes with this DNA under stringent conditions, and has a 6/3 hydroxylase activity of compactin DNA encoding a polypeptide having the formula:

 2. A DNA comprising the nucleotide sequence from positions 544 to 1758 and the nucleotide sequence from positions 1782 to 1970 in the nucleotide sequence of SEQ ID NO: 1, or hybridizing with these DNAs under stringent conditions And a DNA encoding a polypeptide having the 63rd hydroxylase activity of the compactin.

3. In the nucleotide sequence of SEQ ID NO: 1, a polypeptide consisting of an amino acid sequence encoded by DNA having the sequence from position 544 to position 1758, or one or several amino acids are deleted in the amino acid sequence. A polypeptide comprising a substituted or added amino acid sequence and having an activity of the 6 j3 hydroxylase of compactin.

4. In the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 544 to 1758 Thread containing DNA that has a sequence or DNA that hybridizes with this DNA under stringent conditions and encodes a polypeptide having the 6 / 3-position hydroxylase activity of compactin Recombinant DNA.

 5. In the nucleotide sequence of SEQ ID NO: 1, a DNA having the nucleotide sequence from the 544th to the 1758th nucleotide, or a DNA that hybridizes with this DNA under stringent conditions, and a DNA having the nucleotide sequence of SEQ ID NO: 1 A DNA comprising the nucleotide sequence from the 1782-position to the 970-position, or a recombinant DNA comprising the DNA and a DNA that hybridizes under stringent conditions.

6. A microorganism into which the recombinant DNA according to 4 or 5 has been introduced.

7. A microorganism having a DNA having a sequence from the 544th position to the 1758th position in the base sequence represented by SEQ ID NO: 1 described in 1 above, or the DNA under conditions for expressing the polypeptide according to 3 above. Alternatively, culturing the microorganism according to the above 6, into which a recombinant DNA which hybridizes with the DNA under stringent conditions and incorporates a DNA encoding a polypeptide having the activity of 6] 3-hydroxylase of compactin is introduced. And a method for producing and accumulating pravastatin by adding compactin to the culture solution or a solution containing cells separated from the culture solution to cause the reaction to accumulate, and collecting the pravastatin.

8. Under the conditions for expressing the polypeptide according to the above 3, under the conditions of SEQ ID NO: 1 described in the above 2, the sequence from the 544th to the 1758th and the sequence from the 1782th to the 1970th in the nucleotide sequence shown in the above 2 Or a recombinant DNA which hybridizes with the DNA or the DNA under a stringent condition, and which incorporates a DNA encoding a polypeptide having a 6] 3-hydroxylase activity of compactin. Culturing the microorganism according to the above-mentioned 6 in which has been introduced, adding compactin to the culture solution or a solution containing cells separated from the culture solution, and reacting the bacterium to produce and accumulate prapatin. And a method for producing pravastatin. '

9. Streptomyces sp. (Streptomyces sp.) T M-6 (FERM BP-8002) or Streptomyces sp. Streptomyces sp.) T M-7 (FERM BP-8003). Detailed description of the invention

 (1) Microorganism

 In view of the above problems, the present inventor screened a number of microorganisms isolated from soil in Japan. As a result, the following TM-6 and TM-7 strains have a high 6 / 3-position hydroxylating activity of compactin. It has been found that it produces a novel polypeptide.

 Both TM-6 and TM-7 strains form on the oatmeal agar (ISP 3) a bacterium that forms abundant light gray aerial hyphae on the underlying mycelium of brown ash and does not produce soluble pigments. It is.

 For these two strains, the sequence of the 16S rRNA gene was determined as described below 5). As a result, the sequence between the two strains was 100% —the sequence number 4), and these strains were identified as belonging to the genus Streptomyces and Streptomyces sp. TM-6 strains And Streptomyces sp. I ^ treptomyces :) Named TM-7 strain, 1-1-1, Tsukuba-Higashi, Ibaraki, Japan on April 25, 2001 1 Chuo No. 6, Independent Administrative Institution FERM P-18311 and FERM P-18312 deposited at the National Institute of Advanced Industrial Science and Technology, Patent Depositary Depositary, and as of April 5, 2002, International Deposit Nos.FERM BP-8002 and FERM BP-8003 Has been internationally deposited.

Patent No. 2603677 (U.S. Pat. No. 5,179,913) The Streptomyces carbophilus SANK 62585 strain (FERM BP-1145) described in the specification of Japanese Patent No. 2672551 is also similarly used for the 16S rRNA gene. Sequence decision (SEQ ID NO: 6), and the strains were clearly different from those of the TM-6 strain and the TM-7 strain. In addition, the sequence was also determined for Microtetraspora having the hydroxylation activity described in Japanese Patent Application Laid-Open No. 2001-286293 by the present applicant. ■ Recticatena iMicrotetraspora recticatena) IFO 14525 was also used to determine the sequence (SEQ ID NO: 5). However, their homology was low, and they were strains of different genera.

 Figure 1 shows the phylogenetic tree created based on these results.

 (2) DNA analysis

 The cloning of DNΑ (SEQ ID NO: 1) that expresses the 6-position hydroxylase of compactin using the above TM-7 strain will be described.

 The Streptomyces' SPTM-7 strain is cultured, and the obtained strain is disrupted to obtain chromosomal DNA. A PCR reaction is performed on the obtained chromosomal DNA using primers designed from the amino acid sequences of the oxygen-binding region and the heme-binding region that are commonly present in P450 hydroxylase familly. A DNA fragment amplified by the PCR reaction is obtained, and a PCR reaction is further performed based on the DNA fragment to obtain peripheral regions on both sides of the DNA fragment amplified by the first PCR reaction. Thus, DNA involved in the conversion of compactin to pravastatin, which is the object of the present invention, can be obtained.

 The obtained DNA (SEQ ID NO: 1) is composed of a sequence encoding two proteins ^ o xA (sequences 544 to 1758 in the nucleotide sequence of SEQ ID NO: 1) and ox B (SEQ ID NO: 1) , 1782 to 1970).

 The polypeptide encoded by the nucleotide sequence of boxA (positions 544 to 1758) is involved in the 6] -position hydroxylation activity of compactin.

As a result of comparing DNA encoding this polypeptide with DNA encoding various other polypeptides having hydroxylation activity, Streptomyces The homology with the hydroxylase gene (sca = 2 (Gene, 164, 81-85 (1995)) derived from Calpophilus SANK 62585 strain (FERM BP-1145): the amino acid sequence of the enzyme is shown in SEQ ID NO: 9) 75.2%, iMia'otetraspora recticatena) The gene encoding P-450 derived from IFO 14525 (mox A: the amino acid sequence of the enzyme is shown in SEQ ID NO: 10). %, Amycolata autotrophic ^) Gene encoding P-450 at position 25 from N-102 strain (VD25: Amino acid sequence of the enzyme is shown in SEQ ID NO: 11) 45.7% homology with Streptomyces 'lividans-derived hydroxylase gene (the amino acid sequence of the enzyme is shown in SEQ ID NO: 12) and 75.2% homology with Streptomyces' Tendtomyces tendae) Tji901 strain Nikkomycin biosynthetic pathway The homology with the gene encoding pyridyl homoseleonin (monooxykena) ~ pvndvlhomothreonine monooxygenase (Nik F: the amino acid sequence of the enzyme is shown in SEQ ID NO: 13) is 45.8%, indicating high homology. There was no indication.

 Between the polypeptide encoded by the nucleotide sequence of bo XA derived from the TM-7 strain of the present invention and a polypeptide (≤ £ a = 2) derived from Streptomyces cerevisiae strain SANK 62585 (FERM BP-1145) Figure 5 shows the comparison. In the amino acid sequence (single-letter sequence) in the figure, the outlined characters indicate portions where the amino acid sequences match.

Bo XB (1782 to 1970) downstream of bo XA encodes a protein with high homologous 1 "production to ferredoxin, is responsible for electron transfer in boXA's 6-hydroxylation of compactin, and plays an active role in its activity. The enzyme produced by the gene sca-2 of the Streptomyces carpophilus SANK 62585 strain (FERM BP-1145) described in Japanese Patent No. 2603677 and Japanese Patent No. 2672551. Has been shown not to be mediated by ferredoxin, so the enzyme of the present invention is not compatible with sca-2 derived enzyme. Different qualities (Biochimica et Biophysica Acta, 1084 (1991), 35-40).

 (3) Construction of plasmid and preparation of transformant

 (3-1) Preparation of DNA fragment containing both boXA and boxB Based on the nucleotide sequence of SEQ ID NO: 1 analyzed in (2) above,

5. Design and synthesize primers and antisense primers, and prepare primers with appropriate restriction enzyme sites added to the ends of the primers. Specifically 5 'end B g 1 II site the Tsukeryoku tl primers box B gl F (5'-gcagatcttgtgagc gtcgggtgggtaa-3 1: SEQ ID NO: 7) and 5, were added K pn I site at the end Plastic one more bo K n R: may be used (.5 -gcggtaccccgcacggcccctactcgac-3 ¹ SEQ ID NO: 8).

 Next, a PCR reaction is performed using these two primers (boXBg1F and boxKpnR) and chromosomal DNA of TM-7 strain as a template. For the PCR reaction, for example, using Takara LA Taq (Takara Shuzo) and a PCR amplifier (Biometra T Gradient), denaturation is performed at 98 ° C for 20 seconds, annealing and extension are performed at 68 ° C for 2 minutes in two steps. This reaction can be performed under the condition of repeating 25 times.

 As a result, a DNA fragment containing boXA and boxB is amplified, and the DNA fragment is fractionated and recovered by a technique such as agarose electrophoresis.

 (3-2) Construction of plasmid

A plasmid is constructed by a conventional method using the above DNA fragment containing bo XA and box B and a plasmid vector appropriate for the host microorganism. For example, a plasmid vector and the above-mentioned DNA fragment are each digested with the above-mentioned restriction enzymes and ligated using DNA Ligation Kit ver. A plasmid can be constructed in which a DNA fragment containing both the contributing DNAs, bo XA and box B, and a plasmid vector are ligated. . When Streptomyces V. vidans is used as a host microorganism, pIJ702 and pSK117 can be used as the plasmid vector.

 When pIJ702 is used, other host microorganisms such as Streptomyces fradiae and Streptomyces thermotorelans can be used.

 (3-3) Preparation of transformant

 The constructed plasmid is introduced into a host microorganism by a conventional method, and the host microorganism is transformed into a host microorganism, which has bo XA and box B genes. Microorganisms can be obtained.

 There is no particular limitation on the method of introducing the plasmid. For example, when the host microorganism is a microorganism belonging to the genus Streptomyces, a combi- nation cell method or an electroporation method can be employed. The specific method is described in Genetic Manipulation of Streotomyces: A Laboratory Manual. Jonn Innes Foundation, Norwich, 1985 and the like.

 (4) Method for producing pravastatin using microorganisms

A method for culturing the microorganism of the present invention TM-6 or TM-7, or a microorganism transformed with the gene of TM-6 or TM-7 by the above method, and a method for producing pravastatin using compactin as a raw material Will be described. The medium composition used in the present invention includes a carbon source, a nitrogen source, an inorganic salt, and a natural organic material that are suitable for the microorganism to be used to grow well and to express the enzymatic activity of hydroxylating the 6] -position of compactin. It is made up of nutrients. As the carbon source, glucose, fructose, glycerol, sonorebitol, alcoholic acids, acetic acid, starch and the like can be used alone or in combination. The concentration of the carbon source is not particularly limited, and approximately 1 to 10% is appropriate. Nitrogen, ammonia as source, urine One, two or more compounds such as sulfur, ammonium sulfate, ammonium nitrate, and ammonium sulfate can be used. As the inorganic salt, salts such as phosphoric acid phosphate, phosphate phosphate, magnesium sulfate, manganese sulfate and ferrous sulfate can be used. Furthermore, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, etc. are used as organic nutrients that have the effect of promoting the growth of the bacteria used.In addition, small amounts of vitamins and nucleic acids should be included in the medium. Can also.

 The compactin for hydroxylating the 6] -position of compactin may be added either at the beginning of the growth of the cells or after the growth of the cells, or the cells may be recovered from the culture solution and the cells may be added to an appropriate aqueous solution. It may be added to the suspended liquid. It is desirable that the compactin be used at a concentration of about 0.1 g to 2 g / L, which can be used at various concentrations. The compactin may be added at a time or added in portions. Japanese Patent Publication Nos. 62-54476 (U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,489,879), Japanese Patent Publication No. 3-71116 (US Patent No. 4537859), and Japanese Patent Publication No. 7-24579 In the bacterial strain, conversion was performed with the raw material compactin concentration of 0.5 g / L. However, in the TM-6 strain and the TM-7 strain of the present invention, conversion to brapastatin was good even when the raw material compactin concentration was further increased. Therefore, there is an advantage that brapastatin can be efficiently produced with a smaller apparatus.

 The culture may be performed under aerobic conditions such as aeration-agitation culture, shaking culture, or static culture. In culture, H is preferably neutral to weakly alkaline, and known pH neutralizing agents such as ammonia, sodium hydroxide, potassium hydroxide, calcium carbonate, and hydrochloric acid can be used. The cultivation temperature can be in the range of 20 to 40 ° C, and can be cultivated at a temperature optimal for the growth of the used bacteria. The cultivation time varies depending on the strain used, the method of adding the raw materials, the concentration of the added materials, etc., but pravastatin is produced and accumulated in the culture solution in 3 to 10 days.

The produced and accumulated pravastatin can be isolated and purified according to a method known per se. In other words, it absorbs on a hydrophobic carrier or ion-adsorbing resin. It is soaked, eluted with an organic solvent or alkaline water or acidic water, concentrated, and recovered by crystallization. An example of the isolation and purification method is shown below.

 In the culture solution, pravastatin is usually contained as a sodium salt, and can be extracted in a free form under an acidic condition using an extraction solvent such as ethyl acetate or butyl acetate.

 The pretreatment includes: (a) washing the filtrate with butyl acetate or the like under alkaline conditions; or (b) extracting the filtrate with ethyl acetate or the like under the acidic condition, An aqueous solution is added to collect the pravastatin salt in the aqueous layer, and the pravastatin salt is treated with an ion exchange resin and preliminarily purified. Here, in the ion-exchange resin treatment, the pravastatin-containing aqueous layer is neutralized, then immersed in the ion-exchange resin, brapastatin is adsorbed to the resin, and then pravastatin is eluted with, for example, an aqueous solution containing acetone. Do.

 The pravastatin extract is then concentrated. The concentration is preferably performed to such an extent that pravastatin is contained in an amount of 1 O mg or more per lm, although it depends on the concentration of impurities and the like.

 A secondary amine is preferably added to the concentrated extract thus obtained to precipitate a secondary amine salt of brapastatin.

As the secondary amine which forms a salt with pravastatin, R ^ NH (wherein R ¹ and R ² are the same or different alkyl groups having 1 to 8 carbon atoms, cyclic alkyl groups, aryl groups, or substituted A phenyl group which may be used.)) Can be used, but from the viewpoint of the recovery and purity of pravastatin, hexylamine and getylamine are preferred.

 The addition amount of the secondary amine may be 1 equivalent or more with respect to pravastatin. Add the secondary amine and stir at room temperature for one to several hours to precipitate the pravastatin amine salt.

By the above crystallization operation, high purity (absolute purity 99 ~ 100 ° / ₀ ) pravasta Although tin can be obtained in high yield, the amine salt may be further purified by recrystallization. The specific operation of recrystallization depends on the type of amine.

 For example, pravastatin dicycloamine salt is poorly soluble in aqueous solvents and can be purified by recrystallization from an aqueous solvent such as an aqueous alcohol solution. As the recrystallization operation, for example, a hexylamine salt of disocc is dissolved in an aqueous solution of isopropanol while heating, and then allowed to cool to precipitate crystals. After the precipitation, the mixture is preferably cooled to about 0 to 10 ° C. and stirred for about 15 minutes to 1 hour. In addition, in the case of a water-soluble amine salt or ammonium salt other than hexylamine salt, it is purified by recrystallization from an organic solvent. For example, in the case of getylamine, the amine salt is dissolved in methanol, and a non-polar organic solvent such as isopropyl acetate or ethyl acetate is added to precipitate crystals, followed by stirring at room temperature for about 1 to 2 hours. In the conversion reaction of compactin by a microorganism, pravastatin (6β-isomer) and by-produced 6α-isomer, which is an inactive in-vivo epimer, can be removed by recrystallization treatment. In particular, when recrystallized from an aqueous solvent, the 6α-form can be largely removed.

 The amine salt obtained as described above, for example, is suspended in water, adjusted to be acidic with a sulfuric acid aqueous solution or the like, and extracted with an organic solvent such as ethyl acetate to form a free form. Can be. Furthermore, it can be converted to a non-toxic salt such as sodium salt by treating with a sodium hydroxide solution or the like.

As used herein, the term "DNA that hybridizes under stringent conditions" refers to the above-mentioned DNA or a fragment of the DNA as a probe for colony hybridization and plaque hybridization. Or DNA obtained by using the Southern plot hybridization method or the like. Hybridization can be performed according to the method described in Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, 1989, etc. Wear. Specific examples of the hybridizable DNA include DNA having homology of at least 80% or more, preferably DNA having homology of 90% or more with the base sequence such as SEQ ID NO: 1.

 In the present specification, “deletion, substitution or addition of an amino acid” can be performed by site-directed mutagenesis, and “one or several amino acids” It means the number of amino acids that can be deleted, substituted or added by the induction method, for example, 1 to 5 amino acids. A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added can be prepared according to the method described in, for example, the second edition of Molecular Leucacloung. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a phylogenetic tree for gene analysis of the strains of the present invention (TM_6, TM-7) and related strains by the neighbor-joining method. “0.01” on the scale in the figure indicates the degree of genetic proximity.

 FIG. 2 is a graph showing the hydroxylation efficiency at various compactin concentrations by the TM-6 strain and the TM-7 strain of the present invention.

 FIG. 3 shows the results of measuring the change over time in the compactin concentration and the amount of pravastatin produced in Example 3 for each strain by HPLC.

 FIG. 4 shows the results of measuring the time-dependent changes in the compactin concentration and the amount of pravastatin produced in Example 4 for each strain by HPLC.

 FIG. 5 shows the homology of the amino acid sequences of the polypeptides encoded by the present boXA and sca-2.

 FIG. 6 is a HPLC chart of a solution treated with a transformant of the present invention and a microorganism transduced with sca_2.

FIG. 7 shows the results of the transformant of the present invention and the microorganism transduced with —2. It is a graph which shows compactin conversion efficiency. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following description.

Reference example: Preparation of compactin solution

 The compactin was pulverized in a mortar, and the obtained pulverized compactin was dissolved in a 0.2 N aqueous sodium hydroxide solution at high temperature. Hydrochloric acid: neutralized to 7.0 to 7.5 and made up with distilled water to obtain a compactin solution. Example 1:

In a 25 OmL Erlenmeyer flask containing 5 OmL of TSB medium shown in Table 1, the TM-7 strain of the present invention and Japanese Patent Publication No. 62-54476 (U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,489,795) were added. The frozen seeds of the Streptomyces halstedii) IF03199 strain described in the gazette were inoculated at 2 ° / ₀ , respectively, and cultured at 28 ° C and 220 rpm for 24 hours to perform seed culture.

3：6培地5 OmLに種母をそれぞれ 2%植 菌し、 28°C、 2 20 r pmで培養した。 24時間後にサンプリングを行い、 HP LCによりプラバスタチンの生成量 （濃度） およびコンパクチンの残存 量 （濃度） を分析した。 表 1 ： トリプシン大豆 （T S B) 培地 （D co社製） Then 50 at a final concentration of compactin by OmgZL, the F e S0 ₄ ■ 7H ₂ 0 in a final concentration of 1

Seed mothers were each inoculated at 2% in 5 OmL of 3: 6 medium and cultured at 28 ° C. and 220 rpm. Sampling was performed 24 hours later, and the amount of pravastatin produced (concentration) and the amount of remaining compactin (concentration) were analyzed by HP LC. Table 1: Trypsin soybean (TSB) medium (Dco)

 Casein digest 1.7%

 Soy flour papain digest 0.3%

 K, 'Norecos 0.2 5%

 N a C 1 0.5%

K _? HP0 ₄ 0.2 5%

H 7.3 ± 0.2 Further, 5 OmL of the medium shown in Table 2 (the medium used in Example 1 of Japanese Patent Publication No. 62-54476 (U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,489,879)) was used. In a 250-mL L Erlenmeyer flask, the Absidia.bsidia coet'uleci strain IF04423 described in JP-B-62-54476 (U.S. Pat. Nos. 4,346,227, 4,410,629 and 4,489,879) Was inoculated at 2% and cultured at 28 ° C. and 220 rpm for 48 hours to perform seed culture. Subsequently, 2% of the seed mother was inoculated in a 50 OmL volumetric flask containing 10 OmL of the above medium, and 28. C, Cultured at 120 r for 48 hours. After 48 hours, 5 0 0 mg / L final concentration, the _{F e S0 4 · 7 H 2} 0 and 1 0 OmgZL added Caro at a final concentration of compactin was continued for another culture. Sampling was performed 48 hours and 120 hours later, and the amount of pravastatin produced (concentration) and the amount of remaining compactin (concentration) were analyzed by HP LC.

Table 2: Medium composition

 Gunolle course 2%

K ₂ HP0 ₄ 0.1 5%

Mg SO, 7H ₂ 0 0.1 5%

NH ₄ N0 ₃ 0.1%

 Peptone 0.1%

 Corn ■ Steep Li force, 0.2%

 Yeast extract 0.1%

Z n S0 ₄ 7H ₂ 0 0.001 '

 Tap water

 H 7.0 The analysis conditions by HP LC are as follows.

 Shimadzu Corporation CLAS S—VP

Kaka Furamu: YMC-Pack ODS-A A-307 φ 4.6 X 75mm S-5120A

Column temperature 40 ° C

Injection volume 1 μΐ,

Detection wavelength 23 7 nm

Mobile phase liquid A Water: acetic acid: triethyl j-reamine = 998: 1: 1

 Solution B Methanol: acetic acid: triethylamine 2 9 98: 1: 1

： 2.0mL / min

Dalato: 0 minutes B solution 50% → 3 minutes B solution 90% → 3.5 minutes B solution 90% → 3.5 1 minute B solution 50% → 5 minutes 8 solutions

Table 3 shows the results. As is evident from Table 3, the IF03199 strain or the # 4423 strain described in Japanese Patent Publication No. 62-54476 (U.S. Pat.Nos. 4,346,227, 4,410,629 and 4,489,879) produces only pravastatin in the order of several mgZL. However, the TM-7 strain of the present invention produced pravastatin exceeding 20 Omg / L in 24 hours, indicating that it has excellent conversion ability. The conversion was determined from the mass of pravastatin produced relative to the mass of compactin consumed.

 Table 3

実施例 2：

Example 2:

 In a 250 mL flask containing 50 mL of a TSB medium (Difco) shown in Table 1 above, the frozen seeds of the TM-6 strain and the TM-7 strain of the present invention were added at 2 ° / each. The cells were inoculated, cultured at 28 ° C. and 220 rpm for 24 hours, and seed culture was performed.

 Subsequently, MGS medium supplemented with compactin and OJgZL (essan meat 2%, polypeptone 0.5%, sodium glutamate 0.25%, glucose 2%, ferrous sulfate 0.01%, pH unadjusted) Each of the seeds was inoculated in an OmL-volume flask at 2% and cultured at 28 ° C. and 220 rpm.

 After 36 hours of culture, the amount of pravastatin produced was measured by the same HP LC analysis as in Example 1. As a result, pravastatin was detected at 0.25 g / mol in both TM-6 and TM-7 strains.

Furthermore, add 0.5 g / L _N l.Og / compactin to each flask, add 2.0 g / L and 3.0 g / L, and adjust the pravastatin production rate up to 48 hours after HPLC. Was measured by The results are shown in Table 4 and FIG.

 Table 4

表 4およぴ図 2から明らかなように、 0.5〜2.0 g Z Lというかなり高いコン パクチン濃度においても、 本発明の菌株を使用した場合は優れた変換能を維 持しており、また 3.0g/Lという高濃度においても本発明の TM— 6株は優 れた変換能を有していることがわかる。 実施例 3 ：

As is evident from Table 4 and FIG. 2, even at a very high compactin concentration of 0.5 to 2.0 g ZL, excellent conversion ability is maintained when the strain of the present invention is used, and 3.0 g It can be seen that the TM-6 strain of the present invention has excellent conversion ability even at a high concentration of / L. Example 3:

 In a 250 mL Erlenmeyer flask containing three glass beads, 5 OmL of the TSB medium (Dco) shown in Table 1 and the TM-7 strain of the present invention or Patent No. 2603677 (US Patent No. 5179013) 2% of the frozen seeds of Streptomyces carbophylus SANK 62585 strain (FERMBP-1145) described in (1) above, and cultured at 28 ° C and 220 rpm for 24 hours, followed by seed culture. Went.

1/2 impeller comprising subsequently compactin to 5 0 0 μ g / mL final _{concentration, F e S 0 4 · 7} H 2 1 0 0 0 at a final concentration gZmL添Ka卩the TSB 2-fold concentration medium 1.5 L A 3 L minijam fermenter equipped with (6 pieces, 2 pieces, 2 steps, 4.5 cm spacing) is inoculated with 2% of the seed mother and cultured at 28 ° C, 0.5 v vm, 40 Orpm. Ya I

Check after culture 2 2 hours, was 1000μ _g / mL (1500m _g)添力[J compactin, Later, the reduction of compactin concentration by HP LC analysis as shown in Table 5 _T that the appropriate co-添Ka卩in, and the culture was continued

 Table 5

 *: Not added because the concentration has not dropped sufficiently. If the dissolved oxygen (DO) drops, increase the rotation speed of the stirring (up to 700 rpm) and adjust the pH using 50% glucose. The upper limit was adjusted to be within pH 7.5.

 Fig. 3 shows the results of HPLC measurement of the change over time in the compactin concentration and the amount of pravastatin produced for each strain. Similarly, the amount of pravastatin isomer was also measured for the isomer (6 paras) in which the 6a position of the isomer was hydroxylated. Fig. 3 also shows the results.

 As is evident from FIG. 3, the TM-7 strain of the present invention can convert compactin into pravastatin more favorably than the SANK 62585 strain, and the ratio of the amount of pravastatin to the amount of pravastatin produced. Is small. Example 4

 Into a 25 OmL Erlenmeyer flask containing three glass beads, 50 mL of TSB medium (Difco) shown in Table 1 above and 2% of a frozen seed of TM-7 strain of the present invention were inoculated. The cells were cultured at 28 ° C and 220 rpm for 24 hours, and seed culture was performed.

Then 50 at a final concentration of compactin and 0 μ g / mL F e S0 4 · 7H 2 0 3 L mini-jam fermenter equipped with a 1/2 impeller (6 sheets, 6 sheets, 2 steps, 4.5 cm spacing) containing 1.5 L of TSB 2 times concentrated medium supplemented with 100 g of Zm L The seeds were inoculated with 2% and cultured at 28 ° C, 0.5 VVm and 400 rpm.

 After 26 hours of culturing, a compactin solution (25 g / L) was continuously added. The rate of addition was varied as shown in Table 6.

 Table 6

 * Continuous addition starts after 26 hours, and the addition rate is set at each time.

 I am fixing it.

DO, pH, nucleic acids (NA), compactin concentration bra pasta Chin production amount, and Figure 4 shows the production amount of Epima (6 _alpha form).

 As is clear from FIG. 4, it is found that the TM-7 strain of the present invention does not lose its activity even after prolonged culture and accumulates pravastatin up to 4.6 g / L after 160 hours.

Example 5: Sequencing of the 16 S rRNA gene of actinomycetes

In order to carry out a systematic analysis of the actinomycetes TM-6 strain and TM-7 strain of the present invention by the neighborhood binding method, the sequence of the 16S rRNA gene was determined, and the sequence was determined. The genus of microorganisms was identified by analysis of the microorganisms.

 Also, the deposited bacteria of Streptomyces ■ carpophyllus SANK 62585 (FERM BP-1145) described in Patent Nos. 2603677 (US Pat. No. 5,179,913) and 2,672,551 were obtained, and the 16S rRNA gene was similarly obtained. Sequence was determined.

 The operation method is as follows.

 (1) DNA extraction

 DNA extraction from actinomycetes was performed as follows. Actinomycetes cultured in C medium at 30 ° C. and 200 rpm for 2 days were collected by centrifugation at 5000 rpm for 3 minutes to obtain bacterial cells. The cells were resuspended in 360 μL of ΤΕ (1 OmM Tris-HC1 (pH 8.0), ImM EDTA) and freeze-thawed twice. Then, 40 zL of 1 Omg / mL lysozyme (final concentration of 1 mg Zml) was added and incubated at 50 for 30 minutes. Further, 100 μL of 1 Omg / mL protein kinase K (final concentration 1 mgZmL) and 500 μM of BL buffer (Tris — HC 1 (pH 8.0; 4 OmM), Tween 20 (1 %), Nonidet P-40 (0.2%), and EDTA (0.2 mM)), and the mixture was further incubated for 30 minutes. After inactivating protein kinase K by incubation at 90 ° C. for 10 minutes, 1 μL thereof was used for the PCR reaction shown below.

 (2) PCR amplification

The PCR reaction was performed using Takara PCR Thermal Cycle MP (Takara Shuzo). The primer set used for PCR is designed to specifically amplify almost the entire length (9-1541 bases) of small submit liposomal RNA (16S rRNA) gene of eubacteria. For 9F (5, -gtgtttgatcctggctcag (SEQ ID NO: 14)), 1541 R (5'-aaggaggtgatccagcc (SEQ ID NO: 15)) was used instead. PCR Cycle The program was preheated at 96 ° C for 2 minutes, followed by 30 cycles of PCR. 1 cycle The denaturation step was performed at 96 ° C for 20 seconds, the annealing step was 50 ° C for 20 seconds, and the extension step was 72 ° C for 2 minutes.

 After confirming the amount and length of the PCR amplification product obtained by 1% agarose gel electrophoresis, the product is purified with a spin 'column (QIA quick PCR Purification Kit, manufactured by QIAGEN), and the type I DNA for sequencing is purified. And

 (3) Sequence decision

 The sequence was determined without direct cloning by direct sequencing of the PCR amplification product. The sequence was determined at about 1000 bases, which is about 2/3 of the amplified 16 S rRNA gene. As sequence primers, 9 F (5'-gtgtttgatcctggctcag (SEQ ID NO: 14)), 5 10 F (5'-gtgccagcagccgcggt (Toroki self! J No. 16)), 10 9 9 F (5 ' -gcaacgagcgcaaccc (SEQ ID NO: 17)) and 536R (5'-gtattaccgcggctgctg (SEQ ID NO: 18)), and the sequence was determined using an ABI 310 automatic fluorescence sequencer (Perkin-Elmer). The sequencing reaction was carried out using a special kit, Big 'Dye terminator Cycle sequence kit (manufactured by Perkin-Elmer). As a result, the sequence shown in SEQ ID NO: 4 was obtained for the TM-6 strain and the TM-7 strain (100% homology), and the sequence shown in SEQ ID NO: 6 was obtained for Streptomyces' Carpophilus SAN 62585 (FERMBP-1145). The sequence was obtained.

 (4) System analysis

 The system analysis by the neighbor-joining method was performed using the system analysis program PHYL IP o

The previously reported Streptomyces (16SrRNA gene of the Streptomyces group) collected from the sequence data of SEQ ID NO: 4 obtained from the database (GenBank, EMBL, DDBJ, RDP) Program ■ package with sequence, aligned using 1 ustar-1X. The sequence data of TM-7 strain (SEQ ID NO: 4) was compared with the 16S rRNA gene sequence of the Streptomyces treptomyces group at the gene level by the neighbor-joining method. (Fig. 1).

 Based on the above results, the strain TM-6 and the strain TM-7 belong to the genus Streptomyces, but are described in Patent No. 2603677 (US Patent No. 5179013) and Streptomyces carpophilus described in Patent No. 2672551. It was confirmed that this was a different species. These strains were Streptomyces sp. TM-6 (Streptomyces sp. TM-6: Accession number FEM BP-8002) and Streptomyces' SP TM-7 {Streptomyces sp. TM-7: Accession number FERM BP -8003), and deposited internationally with the National Institute of Advanced Industrial Science and Technology on April 5, 2002. Example 6: Determination of nucleotide sequence derived from TM-7 strain

 (1) Preparation of DNA of Streptomyces sp. TM—strain chromosome 7 Glucose 1. The TM-7 strain was inoculated into a medium containing malt extract 0.4% and yeast extract 1%, and cultured at 28 ° C for 3 days. The obtained culture was centrifuged at 3000 rpm for 10 minutes to collect cells. Chromosomal DNA was prepared from the cells using a Blood & Cell Culture kit (QIAGEN).

 (2) Cloning of partial sequence of DNA encoding protein having compactin hydroxylation activity

 Based on the description of Gene, 1, 81-85 (1995), a consensus sequence (Glu Phe Thr Val Lys) between the amino acid sequence of P450 sca-2 and the amino acid sequence of Mo xA (SEQ ID NO: 10) The following mix-primers (P450F2 and P45OR2) were designed and prepared from Arg and His Gin Cys Leu Gly Gin (see SEQ ID NOS: 19 and 20 in the sequence listing).

P450F2: 5'-gagttcacsgtsaagcg-3 ' P450R2: 5'-tgsccsaggcactggtg-3 '

 In order to increase the reactivity in consideration of codon fluctuation, a mixed base s (= c 10 g) was used.

 Next, a PCR reaction was performed using these two primers (P450F2 and P450R2) and the TM-7 strain chromosomal DNA obtained in the above (1) as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifying device (Biometra T Gradient) at 98 ° C for 20 seconds, denaturation at 40 ° C for 2 minutes, elongation at 68 ° C, Performed for 30 seconds. The three-step reaction was repeated 30 times. As a result, a DNA fragment having a size of about 750 bp (hereinafter, DNA fragment 1A) was amplified. This DNA fragment A is likely to be a part of DNA encoding a protein having hydroxylation activity. The reaction solution containing the DNA fragment 1A amplified by the PCR reaction was fractionated by agarose gel electrophoresis. The DNA fragment _A having a size of about 750 bp was cut out from the agarose gel and recovered by SUPREC01 (Takara Shuzo).

Next, in order to obtain a sufficient amount of the DNA fragment 1A to analyze the nucleotide sequence of the obtained DNA fragment 1A, the DNA Ligation kit ver.2 was added to a plasmid vector; T7B1ueT (Novagen). DNA fragment 1A was ligated using (Takara Shuzo) to transform Escherichia coli JM109 strain. Then, ampicillin (50 g / mL), X-ga1 (5-Bromo-4-Chloro-3-Indolyl-j3-D-Galactoside; 40 μg / mL), I PTG (Isopropyl-j3-D-thiogalactopyranoside ; 1 00 Micromax) LB agar medium (1.0% Bata Toto Lipton containing 0.5% yeast _{^{extract, l 0/0 N a C}} l, p H7.0, with 1.5% agar), Escherichia coli transformed Selected. The transformed E. coli colonies thus isolated were cultured in an LB liquid medium (1% batatotryptone, 0.5% yeast extract, 1% NaC1, H7.0) containing ampicillin (50 / gZniL). Separation and purification of plasmid DNA from the transformed transformed E. coli cells using a plasmid purification kit (QIAfilterPlasmidMidiKit, OIAGEN) To obtain a fixed amount of DNA fragment 1A.

 (3) Analysis of nucleotide sequence of cleaved DNA fragment 1A

 The nucleotide sequence of the DNA fragment 1A obtained in the above section (2) was analyzed using a DNA terminator (PE Biosystems 377XL) by the Dye Terminator One Cycle Sequence method. As a result of the nucleotide sequence analysis, the DNA fragment A amplified by the PCR reaction was measured to be about 750 bp by electrophoresis, but the nucleotide sequence analysis revealed that it was 737 bp accurately (sequence No. 1 base 877 to base 1613). At both ends of the cloned 737 bp DNA sequence, DNA sequences corresponding to the two primers used in the PCR reaction were found. It was revealed that A was specifically amplified by these two primers (P450F2 and P450R2).

 (4) Analysis of the peripheral region of DNA fragment A

As described above, since the partial sequence of DNA encoding a protein having hydroxylation activity derived from TM-7 strain was determined, it was determined by the inverse PCR method (Cell Engineering Vol. 14, p.591-593, 1995). We amplified, cloned, and sequenced the base sequence of the peripheral region spreading upstream and downstream of the Clawing fragment. That, TM-7 strain chromosomal DNA ((1) see) the H buffer (50 mM Tr is -HC 1, H7.5, 1 OmM Mg C 1 2, 1 OmM di Chio dithiothreitol, 100m MN a C 1 ) Was digested with restriction enzymes M1uI and PstI, respectively. Each restriction enzyme-cleaved DNA fragment obtained was self-cyclized using DNA Ligation Kit ver.2 (Takara Shuzo).

 On the other hand, the following primers (InvFl and InvRl) were designed and prepared from the base sequence of DNA fragment 1A (see SEQ ID NOS: 21 and 22). InvFl: 5'-ggctccgtcttcgccgacccggacgcctt-3 '

InvRl: 5'-gacgggcagcgcgaactggctgatcaggt-3 ' Next, PCR was carried out using these two primers (In VF1 and InVR1) and the self-cyclized TM-7 strain chromosomal DNA as a template. The PCR reaction was performed using TakaraLATaq (Takara Shuzo Co., Ltd.) and a PCR amplifier (Biometra T Gradient), denaturation at 98 ° C for 20 seconds, annealing and extension at 68 ° C for 4 minutes, and a two-step reaction was repeated 25 times. Was.

 As a result, a DNA fragment having a size of about 7 kbp (DNA fragment 1B) and a DNA fragment having a size of about 6 kbp (DNA fragment-C) were amplified. , And DNA having a DNA sequence containing the upstream and downstream regions thereof.

 This PCR amplification reaction solution was subjected to agarose gel electrophoresis to fractionate. DNA fragments having a size of about 7 kbp and> 6 kbp were cut out from the agarose gel and recovered by SUPREC 01 (Takara Shuzo). Next, for the obtained DNA fragment 1B and DNA fragment 1C, a plasmid vector: pT7B1ue was obtained in the same manner as in the above (2), in order to obtain a sufficient amount of each DNA fragment for nucleotide sequence analysis. Using T (Novagen), DNA Ligation kit ver.2 (Takara Shuzo), E. coli JM109 strain and a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN), a certain amount of each DNA fragment was obtained. Was.

 (5) Analysis of nucleotide sequence of DNA fragment—B (about 7 kbp size) and DNA fragment-C (about 6 kbp size)

The nucleotide sequences of DNA fragment-B and DNA fragment-C obtained in (4) above were analyzed by a DNA terminator one-cycle sequence method using a DNA nucleotide sequence analyzer (PE Biosystems 377XL). The nucleotide sequence was analyzed in this manner, and information on the 1992 bp nucleotide sequence shown in SEQ ID NO: 1 was obtained from the DNA fragment-B and the DNA fragment-C sequence. Example 7:

 (1) Preparation of DNA fragment containing both boXA and boxB from TM-7 strain

And the nucleotide sequence of SEQ ID NO: 1 was analyzed in Example 6 as a reference, 5, primer ends were added to B g 1 II site bo XB g 1 F (5'- gcagatc ttgtgagcgtcgggtgggtaa-3 1: SEQ ID NO: 7) And 5, the n I site was added at the end to design and produce 7 フ フ イ マ b b b ο K pn R (5'-gcggtaccccgcacggcccctactcgac-3 ': J8).

 Next, a PCR reaction was performed using these two primers (boXBg1F and boxKpnR) and the TM-7 strain chromosomal DNA obtained in Example 6 (1) as a template. The PCR reaction was performed using TakaraLATaq (Takara Shuzo Co., Ltd.) and a PCR amplification device (Biometra T Gradient), denaturing at 98 ° C for 20 seconds and annealing and elongation at 68 ° C for 2 minutes. Repeated times.

 As a result, a DNA fragment having a size of about 1.5 kbp and containing boxA and boxB (hereinafter, referred to as DNA fragment-D) was amplified. This PCR amplification reaction solution was subjected to agarose gel electrophoresis to fractionate. The above DNA fragment _D having a size of about l.5 kbp was cut out from the agarose gel and recovered by SUPREC 01 (Takara Shuzo).

 (2) Construction of plasmid p I J bo xAB

pIJ702 was digested with restriction enzymes BgIII and JL ^ iil in T buffer (33 mM Tris-HC1, H7.9, 10 mM magnesium acetate, 0.5 mM dithiothreitol, 66 mM acetate). Digestion yielded a plasmid digest. Similarly, the DNA fragment 1D obtained in the previous section (1) is digested with restriction enzymes BgIII and KpnI, and the digested DNA fragment 1D and the plasmid digest are combined with the DNA Ligation Kit ver. 2 (Takara Shuzo). As a result, boxA and DNA, which are involved in the biological conversion of compactin to pravastatins, have been developed. A plasmid having a size of about 6.5 kbp (hereinafter, referred to as plasmid pIJ bo xAB) was constructed in which a DNA fragment 1D containing both oxB and oxB was ligated to plasmid pIJ702.

 (3) Preparation of Transformant Streptomyces' lividans Streptomyces lividans) p I Jbox AB strain (Streptomyces lividans Streptomyces lividans) p I Jsca 2 strain

 Using the plasmid pIJbo xAB prepared in (2) above, the Streptomyces lividans TK21 strain was transformed according to the method described in the Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, 1985. Converted. Thus, a Streptomyces lividans; pIJbox AB strain transformed with the plasmid pIJboxAB was obtained.

 According to the method described in Gene, 1 ^ (1995), 81-85, the same plasmid as pSCA205 described here was prepared and designated as pIJsca2. Using Plasmid KpIJsca2, the Streptomyces lividans TK21 strain was transformed according to the method described in Genetic Manipulation of Streotomyces: A Laboratory Manual, John Innes Foundation, Norwich, 1985. Thus, a Streptomyces lividans pIJsca2 strain transformed with the plasmid pIJsca2 was obtained.

 (4) Conversion of compactin to pravastatin by transformants

Transformants Streptomyces obtained in the previous section (3) ■ Ribidans p IJ box AB strain, Streptomyces lividans p IJ sca 2 strain, and Streptomyces lividans! The frozen seed of IJ7022 strain was inoculated into 25 mL of TSB medium (Difco) containing 25 μg / mL of thiopeptin, and cultured with shaking at 28 ° C. for 48 hours. 1 mL of the obtained culture was centrifuged (3000 rpm, 5 minutes), and the cells were collected and washed with 25 mM phosphate buffer (ρρ7.0) to obtain washed cells. The washed cells of the pIJ box AB strain obtained in this way are used for the conversion reaction. The suspension was suspended in 1 mL of a buffer (containing 25 mM phosphate buffer (H7.0), 0.2% glycerol, 100 μg / mL ferrous sulfate, and 625 zg / mL compactin). This was reacted at 28 ° C for 7 hours. The reaction solution was extracted with acetonitrile, and the amount of pravastatin was measured by HP LC.

HP LC conditions:

 Shimadzu Corporation CLAS S—VP

Kahum YMC-Pack ODS-A Α-307, φ4.6 X 75mm, S-5, 120A

Column temperature 40 ° C

Detection wavelength 237 nm

Mobile phase: mobile phase A: water: acetic acid: g!) Ethylamine = 100: 0.1: 0.1

 Solution B Methanol: acetic acid: triethylamine = 100: 0.1: 0.1 Flow rate 2.0mL / min

Gradient: start time mobile phase (B)

 0.01 min 50%

 33..00 min 90%

 3.5 min 90%

 3.5 1 minute 50%

 5.0 min 50%

 The results are shown in FIGS. FIG. 6 is a HPLC analysis chart of the solution treated with each transformant, and FIG. 7 is a graph showing the conversion efficiency from compactin to pravastatin.

From these figures, it can be seen that the pravastatin peak at a retention time of 1.8 min, which is not observed in the host strain Streptomyces ■ lividans pIJ7022, was found to be a transformant Streptomyces ′ lividans p IJ box AB strain and Streptomyces lividans : pIJ sca 2 strain confirmed that the substrate compactin (retention time 3.2 min) was reduced. This is box A and bo xB Is involved in the conversion of compactin to pravastatin.

 Furthermore, Streptomyces ■ lividans of the present invention are Streptomyces s. ) It is superior to the IJsca2 strain in the following points.

 (1) The rate of conversion of compactin to pravastatin (the rate of reduction of compactin) is about twice as high.

 (2) There are few 6α-forms. That is, the 6/3 / 6α ratio is Streptomyces ■ lividans: 17.2 for the IJboxAB strain and 7.0 for the Streptomyces-lividans pIJsca2 strain. Examples 8 to 11:

 In Examples 8 to 11, the absolute purity of pravastatin (absolute content of pravastatin (%) in the sample) was determined using the analytical value (calibration curve) of a standard product with a known content. The pravastatin concentration was determined by measuring the pravastatin concentration, and the relative purity was determined by the ratio of the pravastatin peak area to the total peak area detected during the HPLC analysis time of 15 minutes under the following conditions.

 HP LC conditions:

Equipment: CLAS S-VP manufactured by Shimadzu Corporation

Kahum YMC-Pack ODS-A Α-307, φ4.6 X 75mm, S-5, 120A

Force ram temperature 40 ° C,

Detection wavelength 238 nm,

Mobile phase 50% methanol (containing 0.1% each of triethylamine and acetic acid), Flow velocity: Adjusted so that the elution time of pravastatin is 4.6 minutes (about 1.3 mL). Analysis time: 15 minutes. Example 8:

Inoculate lmL of frozen TM-17 strain (FERM BP-8003) in a 250 mL Erlenmeyer flask containing 5 OmL of the TSB medium described in Table 1, and incubate at 28 ° C and 220 rpm for 24 hours. Seed culture was performed. Then compactin at a final concentration of 5 O Omg / L, 2 ° to Tanehaha to 250 m L Erlenmeyer flask containing T SB medium 5 OML where the _{F e S0 4 · 7 H 2} 0 and 100 mg ZL added to a final concentration / _{0 was} inoculated and cultured at 28 ° C and 220 rpm. After 48 hours and 72 hours, compactin was added at a final concentration of 50 OmgZL, and cultivation was performed until 96 hours, to obtain a converted culture solution.

 The conversion reaction culture (3 L) was filtered through celite, adjusted to pH 9.0, and washed with butyl acid (1.27 L). The aqueous layer was adjusted to pH 4.3 with 6mo LZL sulfuric acid, and extracted with ethyl acetate (3.54L). Part of the extract (58 OmL) was concentrated to 30 mL. The content of pravastatin (free form) in the concentrate was 840 mg. Dicyclohexylamine (511 L, 1.3 equivalents) was added thereto, and the mixture was stirred at room temperature for 1.5 hours. The precipitated crystals were filtered and dried to obtain 935 mg of crude crystals (light yellow solid).

 As a result of analysis by HP LC, the relative purity of pravastatin (elution time: 4.6 minutes) was 91.6%, the absolute purity as hexylamine salt in the mouth of dicava was 76%, and the recovery was 59%. The main impurity peaks (elution time (minutes) and their ratio (area%)) were 3.5 minutes (6 epimer form): 1.2%, 5.0 minutes (3α translocation): 0.7%, 12.5 minutes: 0.87%, 13.4 Min: 1.5%. Example 9:

700 mg (532 mg of disc-hexyl hexyl) of the crude crystals obtained in Example 8. Contains amine salts. ) Was dissolved in a 10% aqueous solution of isopropanol (14 mL) by heating, allowed to cool to room temperature, and stirred at 0 ° C for 30 minutes. The precipitated crystals were filtered and dried to obtain 376 mg of crude crystals (white solid). As a result of analysis by HPLC, the relative purity of pravastatin (4.6 minutes) was 98.9%, the absolute purity of dicyclohexylamine salt was 99%, and the recovery was 71%. The main impurity peaks were 3.5 minutes (6 _α- epima integrated): 0.55%, 5.0 minutes (three dislocations): below the detection limit, 12.5 minutes: 0.03%, 13.4 minutes: 0.26%. 6 The α-epima one fell 54%. Example 10:

 Of the crystals obtained in Example 9, lO Omg (containing 99 mg of dicyclohexyl / reamine salt) was dissolved by heating in a 10% aqueous solution of isopropanol (3 mL), and then allowed to cool to room temperature. Stirred at ° C for 15 minutes. The precipitated crystals were filtered and dried to obtain 75.6 mg of crude crystals (white solid). As a result of analysis by HP LC, the relative purity of brapastatin (4.6 minutes) was 99.5%, the absolute purity of dicyclohexylamine salt was 100%, and the recovery was 76%. The analysis values of all the detected impurity peaks were 3.5 minutes (6 hepimers): 0.28%, 8.1 minutes (rataton): 0.07%, 13.4 minutes: 0.11%. 6 α-epimer was reduced by 49%. Example 11:

 The crystals obtained in Example 10 were suspended in 1.5 mL of water and adjusted to pH 4.3 with 1 M sulfuric acid. After extraction with ethyl acetate, an aqueous solution of sodium hydroxide was added so as to have an equimolar amount to pravastatin, and ethyl acetate was further added to obtain 5 Omg of sodium salt of pravastatin. Industrial applicability

Streptomyces ■ Carpophilus SANK 62585 (FERM BP-1145) It is known that hydroxylation at the 6β-position of compactin converts it to pravastatin, but the 6-6 and ΤΜ-7 strains of the present invention have a much higher hydroxylation activity than the above strains. Moreover, compared with the case of using the conventionally known strains, for generating a ratio of separation is Epima body difficult 6 _{alpha-position} is hydroxylated lower separation operation after biosynthesis easily.

 In addition, use microorganisms (transformants) that incorporate DNA that encodes a polypeptide (enzyme) involved in hydroxylation at position 6] of the compactin cloned from strains 6-6 and ΤΜ-7. As a result, pravastatin can be produced more efficiently.

Claims

The scope of the claims 1. Nucleotides 544 to 1758 in the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA containing the rooster sequence and hybridizing with this DNA under stringent conditions, and the activity of 6j3 hydroxylase of compactin DNA encoding a polypeptide having 2. A DNA comprising the nucleotide sequence from positions 544 to 1758 and the nucleotide sequence from 1782 to 1970 in the nucleotide sequence set forth in SEQ ID NO: 1, or a DNA that hybridizes with these DNAs under stringent conditions, and DNA that encodes a polypeptide having a 6/3 hydroxylase activity of Kuching. 3. In the nucleotide sequence represented by SEQ ID NO: 1, a polypeptide consisting of an amino acid sequence encoded by a DNA having the 544th to 1758th rooster sequence, or one or several amino acids in the amino acid sequence described above. A polypeptide comprising a deletion, substitution, or addition of an amino acid sequence, and having a compactin 6-hydroxylase activity. 4. A DNA having the nucleotide sequence from position 544 to position 1758 in the nucleotide sequence shown in SEQ ID NO: 1 or hybridizing with this DNA under stringent conditions, and exhibiting the 6β-hydroxylase activity of compactin. A recombinant DNA into which DNA encoding the polypeptide is incorporated. 5. A DNA having a base sequence from position 544 to position 1758 in the base sequence represented by SEQ ID NO: 1 or a DNA hybridizing with this DNA under stringent conditions, and a base sequence represented by SEQ ID NO: 1 Medium 1782th power DNA containing the nucleotide sequence at position 1970, or a recombinant DNA that incorporates this DNA and DNA that hybridizes under stringent conditions. 6. A microorganism into which the recombinant DNA according to claim 4 or 5 has been introduced. 7. The nucleotide sequence represented by SEQ ID NO: 1 according to Claim 1, under conditions for expressing the polypeptide according to Claim 3, wherein the DNA has the sequence from the 544th position to the 1758th position. Or a recombinant DN which incorporates a DNA that hybridizes with the DNA or its DNA under stringent conditions and encodes a polypeptide having the 6] 3-hydroxylase activity of compactin. The microorganism according to claim 6, into which A has been introduced, is cultured, compactin is added to the culture solution or a solution containing cells separated from the culture solution, and the mixture is reacted to produce and accumulate brapastatin, and collect these. A method for producing pravastatin, comprising: 8. Under the conditions for expressing the polypeptide according to claim 3, in the nucleotide sequence represented by SEQ ID NO: 1 according to claim 2, the sequence from position 544 to position 1758 and the position 1782 A microorganism having the DNA of the sequence from position 1 to position 70, or a polypeptide that hybridizes with the DNA or the DNA under stringent conditions and has the 6] 3-position hydroxylase activity of compactin. The bacterium according to claim 6, wherein the recombinant DNA having the DNA to be incorporated therein is introduced, and compactin is added to the culture solution or a solution containing cells separated from the culture solution, followed by reaction with pravastatin. A method for producing pravastatin, comprising producing and accumulating pravastatin. 9. Streptomyces sp. Streptomyces sp.) TM-6 (FERM BP-8002) or Streptomyces' sp treptomyces sp.) TM-7 (FERMBP-8003).