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WO2002099109A1 - Nouveau polypeptide, adn codant ledit polypeptide et utilisation dudit polypeptide - Google Patents

Nouveau polypeptide, adn codant ledit polypeptide et utilisation dudit polypeptide Download PDF

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Publication number
WO2002099109A1
WO2002099109A1 PCT/JP2002/005252 JP0205252W WO02099109A1 WO 2002099109 A1 WO2002099109 A1 WO 2002099109A1 JP 0205252 W JP0205252 W JP 0205252W WO 02099109 A1 WO02099109 A1 WO 02099109A1
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Prior art keywords
dna
compactin
polypeptide
sequence
seq
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Japanese (ja)
Inventor
Tadashi Fujii
Shinji Hirosue
Yasuhide Aritoku
Tatsuo Morimiya
Osamu Johdo
Kunio Isshiki
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Mercian Corp
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Mercian Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)

Definitions

  • the present invention relates to a novel polypeptide having 6 ⁇ -position hydroxylating activity of compactin, a DNA encoding the polypeptide, and uses thereof.
  • a novel polypeptide that produces brapastatin known as an antihyperlipidemic agent by hydroxylating the ⁇ 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.
  • 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., ⁇ , 1 (1971)).
  • 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.
  • compactin produced by Penicillium citrmum, a kind of blue mold, is known.
  • Compactin has the following formula
  • 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.
  • 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.
  • SANK 62585 strain contains 6 ⁇ -position water of compactin. It describes an enzyme that has oxidase activity and is derived from the microorganism and its gene.
  • 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.
  • 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.
  • the present invention has the following configuration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Streptomyces sp. (Streptomyces sp.) T M-6 (FERM BP-8002) or Streptomyces sp. Streptomyces sp.) T M-7 (FERM BP-8003).
  • 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.
  • 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.
  • TM-7 strain 1-1-1, Tsukuba-Higashi, Ibaraki, Japan on April 25, 2001 1 Chuo No.
  • 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
  • the strains were clearly different from those of the TM-6 strain and the TM-7 strain.
  • 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.
  • 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.
  • 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).
  • polypeptide encoded by the nucleotide sequence of boxA (positions 544 to 1758) is involved in the 6] -position hydroxylation activity of compactin.
  • 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.
  • FERM BP-1145 Streptomyces carpophilus SANK 62585 strain
  • Different qualities Biochimica et Biophysica Acta, 1084 (1991), 35-40).
  • a PCR reaction is performed using these two primers (boXBg1F and boxKpnR) and chromosomal DNA of TM-7 strain as a template.
  • boXBg1F and boxKpnR primers
  • chromosomal DNA of TM-7 strain chromosomal DNA of TM-7 strain.
  • 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.
  • a DNA fragment containing boXA and boxB is amplified, and the DNA fragment is fractionated and recovered by a technique such as agarose electrophoresis.
  • 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.
  • 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.
  • pIJ702 and pSK117 can be used as the plasmid vector.
  • pIJ702 When pIJ702 is used, other host microorganisms such as Streptomyces fradiae and Streptomyces thermotorelans can be used.
  • 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.
  • the method of introducing the plasmid there is no particular limitation on the method of introducing the plasmid.
  • 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.
  • 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.
  • 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.
  • the inorganic salt salts such as phosphoric acid phosphate, phosphate phosphate, magnesium sulfate, manganese sulfate and ferrous sulfate can be used.
  • 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.
  • the culture may be performed under aerobic conditions such as aeration-agitation culture, shaking culture, or static 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.
  • 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.
  • 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.
  • 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.
  • 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.)
  • 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.
  • 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.
  • 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.
  • the amine salt may be further purified by recrystallization.
  • the specific operation of recrystallization depends on the type of amine.
  • 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.
  • an aqueous solvent such as an aqueous alcohol solution.
  • 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.
  • a water-soluble amine salt or ammonium salt other than hexylamine salt it is purified by recrystallization from an organic solvent.
  • 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.
  • a non-polar organic solvent such as isopropyl acetate or ethyl acetate
  • pravastatin (6 ⁇ -isomer) and by-produced 6 ⁇ -isomer which is an inactive in-vivo epimer, can be removed by recrystallization treatment.
  • the 6 ⁇ -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.
  • 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.
  • “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.
  • TM-7 strain of the present invention 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.
  • Table 3 shows the results.
  • 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.
  • 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.
  • 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.
  • 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)
  • OJgZL essan meat 2%, polypeptone 0.5%, sodium glutamate 0.25%, glucose 2%, ferrous sulfate 0.01%, pH unadjusted
  • pravastatin was detected at 0.25 g / mol in both TM-6 and TM-7 strains.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the operation method is as follows.
  • 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 ⁇ L of ⁇ (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.
  • 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.
  • 9F 5, -gtgtttgatcctggctcag (SEQ ID NO: 14)
  • 1541 R 5'-aaggaggtgatccagcc (SEQ ID NO: 15)
  • 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.
  • the product 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.
  • a spin 'column QIA quick PCR Purification Kit, manufactured by QIAGEN
  • 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.
  • sequence primers 9 F (5'-gtgtttgatcctggctcag (SEQ ID NO: 14)), 5 10 F (5'-gtgccagcagccgcggt (Toroki self! J No.
  • 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.
  • 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).
  • 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
  • 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).
  • P450F2 5'-gagttcacsgtsaagcg-3 '
  • P450R2 5'-tgsccsaggcactggtg-3 '
  • 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.
  • 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).
  • 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.
  • ampicillin 50 g / mL
  • X-ga1 (5-Bromo-4-Chloro-3-Indolyl-j3-D-Galactoside; 40 ⁇ g / 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
  • 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.
  • a plasmid purification kit QIAfilterPlasmidMidiKit, OIAGEN
  • 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.
  • 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).
  • 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).
  • 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
  • restriction enzymes M1uI and PstI were 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).
  • InvRl 5'-gacgggcagcgcgaactggctgatcaggt-3 '
  • 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.
  • TakaraLATaq TakaraLATaq
  • a PCR amplifier Biometra T Gradient
  • DNA fragment 1B a DNA fragment having a size of about 7 kbp
  • DNA fragment-C a DNA fragment having a size of about 6 kbp
  • 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).
  • 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.
  • T Novagen
  • DNA Ligation kit ver.2 (Takara Shuzo)
  • E. coli JM109 strain and a plasmid purification kit QIAfilter Plasmid Midi Kit, QIAGEN
  • 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 Example 7:
  • 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 ⁇ ⁇ ⁇ ⁇ b b ⁇ K pn R (5'-gcggtaccccgcacggccctactcgac-3 ': J8).
  • 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.
  • DNA fragment-D 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).
  • 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.
  • 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).
  • boxA and DNA which are involved in the biological conversion of compactin to pravastatins, have been developed.
  • Plasmid pIJ bo xAB 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.
  • 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.
  • the same plasmid as pSCA205 described here was prepared and designated as pIJsca2.
  • 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.
  • a Streptomyces lividans pIJsca2 strain transformed with the plasmid pIJsca2 was obtained.
  • 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 ⁇ g / 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.
  • FIG. 6 is a HPLC analysis chart of the solution treated with each transformant
  • FIG. 7 is a graph showing the conversion efficiency from compactin to pravastatin.
  • 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.
  • Streptomyces ⁇ lividans of the present invention are Streptomyces s. ) It is superior to the IJsca2 strain in the following points.
  • the rate of conversion of compactin to pravastatin (the rate of reduction of compactin) is about twice as high.
  • 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.
  • 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).
  • Example 10 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.
  • Streptomyces ⁇ Carpophilus SANK 62585 (FERM BP-1145) It is known that hydroxylation at the 6 ⁇ -position of compactin converts it to pravastatin, but the 6-6 and ⁇ -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.
  • microorganisms that incorporate DNA that encodes a polypeptide (enzyme) involved in hydroxylation at position 6] of the compactin cloned from strains 6-6 and ⁇ -7.
  • pravastatin can be produced more efficiently.

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Abstract

L'invention concerne un nouveau micro-organisme Streptomyces sp. qui produit de la pravastatine (médicament utilisé pour lutter contre l'hyperlipidémie) par hydroxylation de compactine en position 6β, ainsi qu'un nouveau polypeptide produit par le micro-organisme précité. L'invention concerne par ailleurs un ADN codant ledit polypeptide, un ADN recombiné obtenu par intégration de l'ADN précité, un micro-organisme comportant l'ADN précité transféré en lui, ainsi qu'un procédé de production de pravastatine à l'aide du micro-organisme précité.
PCT/JP2002/005252 2001-06-01 2002-05-30 Nouveau polypeptide, adn codant ledit polypeptide et utilisation dudit polypeptide Ceased WO2002099109A1 (fr)

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JP2003026634A (ja) * 2001-07-17 2003-01-29 Mercian Corp プラバスタチンナトリウム塩の製造方法
JP2008521448A (ja) * 2004-12-03 2008-06-26 テバ ジョジセルジャール ザ−トケルエン ムケド レ−スベニュタ−ルシャシャ−グ コンパクチン水酸化能を有する株を構築する方法
WO2010041619A1 (fr) 2008-10-06 2010-04-15 メルシャン株式会社 Vecteur d'expression pour pseudonocardia autotrophica
JP2010512733A (ja) * 2006-12-13 2010-04-30 ディーエスエム アイピー アセッツ ビー．ブイ． プラバスタチンを調製するための方法
US8058037B2 (en) 2005-11-29 2011-11-15 Kyowa Hakko Bio Co., Ltd. Protein and DNA encoding the protein
US8637249B2 (en) 2008-11-14 2014-01-28 Gen-Probe Incorporated Compositions, kits and methods for detection of Campylobacter nucleic acid

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003026634A (ja) * 2001-07-17 2003-01-29 Mercian Corp プラバスタチンナトリウム塩の製造方法
JP2008521448A (ja) * 2004-12-03 2008-06-26 テバ ジョジセルジャール ザ−トケルエン ムケド レ−スベニュタ−ルシャシャ−グ コンパクチン水酸化能を有する株を構築する方法
US8058037B2 (en) 2005-11-29 2011-11-15 Kyowa Hakko Bio Co., Ltd. Protein and DNA encoding the protein
JP2010512733A (ja) * 2006-12-13 2010-04-30 ディーエスエム アイピー アセッツ ビー．ブイ． プラバスタチンを調製するための方法
WO2010041619A1 (fr) 2008-10-06 2010-04-15 メルシャン株式会社 Vecteur d'expression pour pseudonocardia autotrophica
JP5504521B2 (ja) * 2008-10-06 2014-05-28 日本マイクロバイオファーマ株式会社 シュードノカルディア・オートトロフィカ（Ｐｓｅｕｄｏｎｏｃａｒｄｉａａｕｔｏｔｒｏｐｈｉｃａ）発現ベクター
US9006412B2 (en) 2008-10-06 2015-04-14 Microbiopharm Japan Co., Ltd. Expression vector for pseudonocardia autotrophica
US8637249B2 (en) 2008-11-14 2014-01-28 Gen-Probe Incorporated Compositions, kits and methods for detection of Campylobacter nucleic acid
US9175353B2 (en) 2008-11-14 2015-11-03 Gen-Probe Incorporated Compositions, kits and methods for detection of campylobacter nucleic acid
US10829824B2 (en) 2008-11-14 2020-11-10 Gen-Probe Incorporated Compositions, kits and methods for detection of campylobacter nucleic acid

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