WO2012029811A1 - リベロマイシンaまたはその合成中間体の製造法、スピロケタール環含有化合物の製造方法、並びに新規抗癌剤、抗真菌剤および骨疾患治療剤 - Google Patents
リベロマイシンaまたはその合成中間体の製造法、スピロケタール環含有化合物の製造方法、並びに新規抗癌剤、抗真菌剤および骨疾患治療剤 Download PDFInfo
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- 0 *[C@](C(C=CC(*)=CCC(C(*)CC1)OC1(CCC1*)O[C@@]1C=CC(*)=CC(O)=O)O)C=CC(O*)=O Chemical compound *[C@](C(C=CC(*)=CCC(C(*)CC1)OC1(CCC1*)O[C@@]1C=CC(*)=CC(O)=O)O)C=CC(O*)=O 0.000 description 2
- RGZZOCFMWDYFNS-RWZXQFDVSA-N CCCCC(CC1)C(/C=C/C(/C)=C/CC(CC)=O)O[C@@H]1OC(C/C=C(\C)/C=C/C(C(C)/C=C/C(O)=O)O)C(C)CC Chemical compound CCCCC(CC1)C(/C=C/C(/C)=C/CC(CC)=O)O[C@@H]1OC(C/C=C(\C)/C=C/C(C(C)/C=C/C(O)=O)O)C(C)CC RGZZOCFMWDYFNS-RWZXQFDVSA-N 0.000 description 1
- RLKCXRQUMCYPRL-ZRSZQKHOSA-N CCCCC1C(C)O[C@H](CCC(C)C(C/C=C(\C)/C=C/C(C(C)/C=C/C(O)=O)O)O)CC1 Chemical compound CCCCC1C(C)O[C@H](CCC(C)C(C/C=C(\C)/C=C/C(C(C)/C=C/C(O)=O)O)O)CC1 RLKCXRQUMCYPRL-ZRSZQKHOSA-N 0.000 description 1
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Definitions
- the present invention relates to a novel bacterium for producing reveromycin A or a synthetic intermediate thereof using genetic recombination, a method for producing reveromycin A or a synthetic intermediate using the same, and a method for producing a spiroketal ring-containing compound. is there.
- the present invention also relates to a novel anticancer agent, antifungal agent, and bone disease therapeutic agent.
- Reveromycin A is known to induce apoptosis at low concentrations selectively in osteoclasts and to suppress bone metastasis of tumors (Patent Document 1).
- RM-A is a polyketide compound having a spiroketal ring, and a synthetic chemical method has been established, but a multi-step synthesis step is required.
- SN-593 Streptomyces spp.
- Patent Document 2 it is difficult to produce in large quantities. Since no gene involved in reveromycin biosynthesis has been reported so far, reveromycin-producing bacteria have not been bred by genetic recombination.
- JP 7-223945 A Japanese Patent Laid-Open No. 4-49296
- the present inventors have intensively studied to solve the above problems. As a result, the present inventors have succeeded in identifying a reveromycin biosynthetic gene cluster, and found that Streptomyces bacteria in which the revQ gene is highly expressed efficiently produce reveromycin A and its synthetic intermediate. Furthermore, the revG and revJ gene products have been found to catalyze the formation reaction of spiroketal rings, and it has been found that spiroketal ring-containing compounds can be efficiently produced by using this. Furthermore, it has been found that the compounds represented by the following general formulas (III) and (IV) are effective as anticancer agents, antifungal agents, and bone disease therapeutic agents. Based on the above findings, the present invention has been completed.
- the present invention is as follows.
- a revQ encoding a Streptomyces bacterium having the ability to produce reveromycin A or a synthetic intermediate thereof, and encoding the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having 80% or more identity with the amino acid sequence A Streptomyces bacterium modified so that gene expression is increased as compared with the parent strain, whereby the production ability is improved as compared with the parent strain.
- [3] The bacterium according to [1] or [2], which is obtained by modifying Streptomyces sp. SN-593 strain so that expression of revQ gene is increased.
- the Streptomyces bacterium according to any one of [1] to [3] is cultured in a medium, and reveromycin A or a synthetic intermediate thereof is accumulated in the medium.
- RevG protein having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 80% or more identity with the amino acid sequence is allowed to act on the following compound (I) to thereby convert the compound (I) into the following compound (II
- R 1 and R 3 are independently hydrogen or a saturated or unsaturated aliphatic hydrocarbon group having 1 to 25 carbon atoms (the hydrogen atom is substituted with a hydroxyl group, a carboxyl group, an oxo group, a phenyl group or a pyridyl group) And two hydrogen atoms may form a ring by —O—), and R 2 and R 4 are each independently an alkyl group having 1 to 10 carbon atoms.
- R 2 and R 4 are each independently an alkyl group having 1 to 10 carbon atoms.
- An anticancer agent comprising a compound represented by the following general formula (III) or (IV) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 5 , R 6 , R 7 , R 8 and R 9 each represent an alkyl having 1 to 6 carbon atoms
- R 10 represents a hydrogen atom or an alkyl having 1 to 5 carbon atoms.
- R 11 , R 12 , R 13 , R 14 and R 15 are alkyl having 1 to 6 carbon atoms.
- An antifungal agent comprising a compound represented by the following general formula (III) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 5 , R 6 , R 7 , R 8 and R 9 represent alkyl having 1 to 6 carbon atoms
- R 10 represents a hydrogen atom.
- a bone disease therapeutic agent comprising a compound represented by the following general formula (IV) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 11 , R 12 , R 13 , R 14 and R 15 are alkyl having 1 to 6 carbon atoms.
- reveromycin A and its synthetic intermediate can be produced efficiently.
- a spiroketal ring-containing compound can be produced efficiently.
- novel anticancer agents, antifungal agents, and bone disease therapeutic agents can be provided.
- Map of 140-kb region shows contig information of whole genome shotgun sequence, KS-AT ( ⁇ ) and ER ( ⁇ ) sequences used for RT-PCR, and seven fosmid clones (10E09, 11A02, 16E02, 16F06, 18G01, 23G06 and 30B04). The gene region predicted to be involved in reveromycin biosynthesis is indicated by a bold line.
- revQ gene disruption scheme A
- confirmation of revQ gene disruption B
- metabolite analysis results C: wild strain
- D revQ disrupted strain
- E revQ introduced into revQ disrupted strain
- F RevQ overexpressing strain in the wild strain
- LC-MS analysis of RevG reaction products (A) shows the HPLC profile after 0, 1, 5 and 10 minutes.
- (b, c, d) ⁇ Substrate from 0 min sample (RM-A1a: Fig. 2 (14))
- Product from 1 min sample (RM-A2a: Fig. 2 (17)
- 10 min 2 shows mass spectrometry results of products (RM-A3a, A3b: (18a) and (18b) in FIG.
- the bacterium of the present invention is a Streptomyces bacterium having the ability to produce reveromycin A or a synthetic intermediate thereof.
- the amino acid sequence of SEQ ID NO: 36 or an revQ gene encoding an amino acid sequence having 80% or more identity with the amino acid sequence is modified so that the expression of the revQ gene is increased compared to the parent strain, whereby the production ability is It is an improved Streptomyces bacterium.
- the biosynthetic pathway of reveromycin A clarified in the present invention is shown in FIG. 2, and examples of reveromycin A synthesis intermediates include RM-A1a, RM-A2a, RM-A3a, RM-A3b, and RM-T. Is mentioned.
- the revQ gene is a polynucleotide that improves the production ability when introduced into a Streptomyces bacterium having the ability to produce reveromycin A or a synthetic intermediate thereof such as Streptomyces sp.
- a gene derived from Streptomyces sp. SN-593 strain having the nucleotide sequence of SEQ ID NO: 35 of 121-951 can be mentioned.
- the revQ gene may be a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the above base sequence as long as it is a gene that improves the ability to produce reveromycin A or a synthetic intermediate thereof.
- stringent conditions it corresponds to 60 ° C., 1 ⁇ SSC, 0.1% SDS, preferably 0.1 ⁇ SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization.
- the conditions for hybridizing at a salt concentration are included.
- the revQ gene encodes an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 36, and reveromycin A Alternatively, it may be a gene that improves the production ability of the synthetic intermediate. Further, the revQ gene encodes an amino acid sequence having a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 36, and production of reveromycin A or a synthetic intermediate thereof It may be a gene that improves performance. Here, one or several means preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.
- RevQ genes derived from Streptomyces bacteria other than Streptomyces sp. SN-593 or other microorganisms can also be used.
- revQ genes genes that improve the ability to produce reveromycin A or synthetic intermediates thereof based on homology can be isolated from the chromosomes of microorganisms, animals and plants, and the nucleotide sequences thereof can be used.
- a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the ORF portion by a hybridization method or a PCR method.
- Modified so that the expression of revQ gene is increased compared to the parent strain means that the expression level of revQ gene is higher than that of the parent strain (pre-modification strain) such as Streptomyces sp. SN-593 It is preferably 1.5 times or more, more preferably 2 times or more per unit cell weight.
- the expression level of the gene can be confirmed by RT-PCR or Northern blotting.
- Streptomyces sp. SN-593 strain was established on June 5, 1990 at the National Institute of Microbiology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Address 305-8566 East of Tsukuba City, Ibaraki Prefecture 1-chome, 1-address, 1-center, 6), the deposit number is FERM P-11503, and after that, it has been transferred to the international deposit, and the deposit number is FERM BP-3406 (Japanese Patent Laid-Open No. 05-051303) .
- the bacterium used in the present invention is obtained by using a bacterium exemplified below as a parent strain in addition to Streptomyces sp. SN-593, and modifying the parent strain so that expression of the revQ gene is increased. be able to.
- the bacterium used as a parent strain in the present invention is not limited to a wild strain, but a mutant strain obtained by a normal mutation treatment such as UV irradiation or NTG treatment, or a genetic technique such as cell fusion or gene recombination. Any strain such as a recombinant strain to be induced may be used.
- the enhancement of the expression of the revQ gene can be performed by a gene recombination method, for example, by increasing the copy number of the revQ gene or replacing the promoter of this gene.
- the revQ gene as described above may be incorporated into a plasmid capable of functioning in the host microorganism so that it can be expressed, and then introduced into the host microorganism.
- the plasmid vector into which the revQ gene can be incorporated is not particularly limited as long as it contains at least a gene that controls the replication growth function in the host bacterium. Examples of the vector involved in the autonomous replication include plasmid DNA, virus, bacteriophage, bacterial chromosome and the like.
- plasmid DNA examples include plasmids derived from E. coli (ColE plasmids such as pBR322, pUC18, pUC19, pUC118, pUC119, and pBluescript). Moreover, the following actinomycete-derived plasmids can also be used.
- pIJ486 Mol. Gen. Genet. 203, 468-478, 1986
- pKC1064 Gene 103, 97-99 (1991)
- pUWL-KS Gene 165,149-150 (1995)
- pIJ702 J. Gen. Microbiol.
- phage DNA examples include ⁇ phage (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11) and the like.
- Introduction of the recombinant DNA into the host can be performed by a known method.
- a method for transforming Streptomyces bacteria lysozyme is used to spheroplast Streptomyces bacteria, and then the recombinant DNA vector and a buffer containing polyethylene glycol are added to the vector.
- Incorporation into cells [Thompson, CJ, et al. (1982) J. Bacteriol., 151, 668-677 or Hopwood, DA, et al. (1985) "Genetic Manipulation of Streptomyces: A Laboratory Manual", The John Innes Foundation, Norwich] is often used.
- it can also carry out by the electric pulse method (Res.
- Confirmation of the introduction of DNA may be performed using a selectable marker gene (for example, ampicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, etc.).
- a selectable marker gene for example, ampicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, etc.
- the expression enhancement of the revQ gene can also be performed by making multiple copies of the revQ gene on the chromosome by a known homologous recombination method.
- the expression enhancement of the revQ gene can also be performed by replacing or modifying the promoter of the revQ gene on the host chromosome. Examples of the promoter replacement method include a known homologous recombination method and a method using a sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73).
- the promoter for expressing the revQ gene or the promoter used to replace the promoter of the revQ gene on the chromosome in the introduction by the above recombinant plasmid or homologous recombination on the chromosome is one that can function in the host bacterium. Although there will be no restriction
- TipA promoter (Gene 94: 53-59, 1990; Gene 103: 97-99, 1991; Gene 166: 133-137, 1995), transcription induced by the addition of antibiotic thiostrepton, glyCAB induced by glycerol Promoters (Mol. Microbiol.
- DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual", Second Edition ", Cold Spring Harbor Laboratory Press, (1989).
- Reveromycin A and its synthetic intermediate are produced by inoculating the above-mentioned bacteria into a nutrient source-containing medium and culturing aerobically.
- the medium used for the culture may be a medium containing a nutrient source that can be used by Streptomyces bacteria, and various synthetic media, semi-synthetic media, natural media, and the like can be used.
- a nutrient source that can be used by Streptomyces bacteria
- various synthetic media, semi-synthetic media, natural media, and the like can be used.
- glucose as a carbon source sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, organic acid, etc.
- organic media such as pharmamedia, peptone, meat extract, yeast extract, soybean flour, casein, amino acid and urea
- inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination.
- Sodium salts, potassium salts, magnesium salts, phosphates, and other heavy metal salts may be added and used as necessary.
- various known antifoaming agents such as Adecanol (registered trademark) and silicone oil can be added to the medium as appropriate, but the addition does not adversely affect the production of the target substance. Need to be. For example, it is preferable to use at 0.5% or less.
- the pH of the medium is preferably in the optimum pH range of microorganisms, usually near neutral.
- the medium temperature should be kept at a temperature at which microorganisms grow well, usually 20 to 40 ° C., particularly preferably around 27 ° C.
- the culture time is generally about 1 to 5 days, preferably about 72 hours.
- reveromycin A and its synthetic intermediate are produced and accumulated.
- the various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, and the like, and the optimum conditions are selected and adjusted from the above ranges according to each.
- Isolation of reveromycin A and its synthetic intermediate produced by the above-mentioned culture is performed by means of utilizing the difference in solubility between reveromycin A or its synthetic intermediate and impurities, means utilizing the difference in adsorption affinity, and difference in molecular weight. These methods can be carried out by any of these means, and each method is used alone, in appropriate combination, or repeatedly.
- reveromycin A since most of reveromycin A is present in the culture filtrate, when the culture filtrate is purified by a combination of various gel filtration chromatography, adsorption chromatography, liquid chromatography, etc., reveromycin A and A fraction containing other active ingredients is obtained. If the powder obtained by freeze-drying this fraction is further purified by development in a system of 18% methanol: 0.01% ammonia, for example, using high performance liquid chromatography (for example, a capsule pack column), A purified white powder can be obtained (Japanese Patent Laid-Open No. 05-051303). Other reveromycin A synthetic intermediates can be obtained by the methods described in the examples.
- the present invention also comprises causing RevG protein having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 80% or more identity to the following compound (I)
- a method for producing compound (II) which comprises the step of converting the compound (I) into the following compound (II).
- R 1 and R 3 are independently hydrogen or a saturated or unsaturated aliphatic hydrocarbon group having 1 to 25 (preferably 1 to 10) carbon atoms (the hydrogen atom is a hydroxyl group, a carboxyl group, R 2 and R 4 may be independently substituted with an oxo group, a phenyl group or a pyridyl group, and two hydrogen atoms may form a ring with —O—. And preferably an alkyl group having 1 to 6 carbon atoms.
- the compound described in the following (i) is preferable as the compound (I), and the compound described in the following (ii) is preferable as the compound (II).
- examples of the compound (I) include the compounds shown in FIG.
- RevG protein examples include a protein derived from Streptomyces sp. SN-593 having the amino acid sequence of SEQ ID NO: 16, but 80% or more, preferably 90% or more, more preferably the amino acid sequence of SEQ ID NO: 16. It may be a protein having an identity of 95% or more, particularly preferably 99% or more and having an activity of catalyzing a reaction for converting the compound (I) into the compound (II). Further, the RevG protein has a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 16, and a reaction for converting Compound (I) into Compound (II). It may be a protein having a catalytic activity.
- one or several means preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.
- the RevJ protein include a protein derived from Streptomyces sp. SN-593 having the amino acid sequence of SEQ ID NO: 20, and 80% or more, preferably 90% or more, more preferably the amino acid sequence of SEQ ID NO: 20. It may be a protein having an identity of 95% or more, particularly preferably 99% or more and having an activity of catalyzing a reaction of converting Compound (I) to Compound (II) together with RevG.
- the RevJ protein has a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 20, and converts Rev (G) into Compound (II) together with RevG. It may be a protein having an activity of catalyzing the reaction. Here, one or several is the same as described above.
- RevG protein and RevJ protein can be obtained by purification from Streptomyces bacteria such as Streptomyces sp. SN-593
- the revG gene and RevJ protein can be obtained from a suitable host (Streptomyces bacteria, Escherichia It can also be obtained by purifying RevG protein and RevJ protein after expression in cell-free system (genus bacteria, yeast, etc.). At this time, for ease of purification, it is preferable to express by fusing a tag such as polyhistidine or GST and using the affinity for the tag.
- the revG gene that can be used in this case is not particularly limited as long as it encodes a protein having an activity of catalyzing the reaction of converting compound (I) to compound (II).
- 121-939 of SEQ ID NO: 13 A gene derived from Streptomyces sp. SN-593 strain having a base sequence can be mentioned.
- the revJ gene is not particularly limited as long as it encodes a protein having an activity of catalyzing the reaction of converting compound (I) into compound (II) together with RevG.
- a revJ gene has a base sequence of 121-1128 of SEQ ID NO: Examples include genes derived from Myces sp. ⁇ ⁇ SN-593 strain.
- the revG gene and the revJ gene are stringent with DNA having a complementary sequence of the above base sequence as long as they encode a protein having an activity of catalyzing the reaction of converting compound (I) into compound (II). It may be DNA that hybridizes under conditions.
- stringent conditions it corresponds to 60 ° C., 1 ⁇ SSC, 0.1% SDS, preferably 0.1 ⁇ SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization.
- the conditions for hybridizing at a salt concentration are included.
- RevG protein and RevJ protein may be used.
- a fraction containing the RevG protein and RevJ protein, crude purification, or bacteria may be used.
- the reaction of the RevG protein, or RevG protein and RevJ protein with the compound (I) may be performed by mixing both in a solution.
- the reaction temperature is a temperature suitable for the enzyme reaction, but is preferably 20 to 40 ° C.
- revC gene SEQ ID NO: 1 (the encoded amino acid sequence is SEQ ID NO: 2)
- revA gene SEQ ID NO: 3 (the encoded amino acid sequence is SEQ ID NO: 4)
- revB gene SEQ ID NO: 5 (the encoded amino acid sequence is SEQ ID NO: 6)
- revD gene SEQ ID NO: 7 (the encoded amino acid sequence is SEQ ID NO: 8)
- revE gene 121-1221 of SEQ ID NO: 9 (the encoded amino acid sequence is SEQ ID NO: 10)
- revF gene 121-1569 of SEQ ID NO: 11 (the encoded amino acid sequence is SEQ ID NO: 12) revH gene: 121-1641 of SEQ ID NO: 15 (the encoded amino acid sequence is SEQ ID NO: 16) revI gene: 121-1311 of SEQ ID NO: 17 (the encoded amino acid sequence is SEQ ID NO: 18 (the encoded amino acid sequence is SEQ ID NO:
- Each gene described above is a DNA that hybridizes under stringent conditions with a complementary strand of each base sequence as long as it encodes a protein having an activity involved in reveromycin biosynthesis as shown in the Examples. May be.
- each gene described above encodes a protein having an activity involved in reveromycin biosynthesis as shown in the examples, one or several amino acids at one or more positions in each amino acid sequence. It may be a homologue, mutant or artificial modification that encodes an amino acid sequence including substitution, deletion, insertion or addition.
- “one or several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 1 to 20, preferably 1 to 10, more preferably 1 to Mean 5.
- each gene described above is 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97%, based on the entire amino acid sequence, as long as it encodes a protein involved in reveromycin biosynthesis. It may be a gene encoding an amino acid sequence having% or more identity.
- the anticancer agent of the present invention contains a compound represented by the following general formula (III) or (IV) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 5 , R 6 , R 7 , R 8 and R 9 represent alkyl having 1 to 6 carbon atoms, and R 5 , R 7 , R 8 and R 9 are preferably alkyl having 1 to 3 carbon atoms, and more Preferably it is methyl.
- R 6 is preferably alkyl having 3 to 6 carbon atoms, and more preferably butyl.
- R 10 represents a hydrogen atom or alkyl having 1 to 5 carbon atoms, preferably a hydrogen atom, methyl or ethyl.
- R 11 , R 12 , R 13 , R 14 and R 15 represent alkyl having 1 to 6 carbon atoms
- R 11 , R 13 , R 14 and R 15 are preferably alkyl having 1 to 3 carbon atoms, and more Preferably it is methyl.
- R 12 is preferably alkyl having 3 to 6 carbon atoms, and more preferably butyl.
- Examples of the pharmaceutically acceptable salt include mineral acid salts such as hydrochloride and sulfate; or organic acid salts such as p-toluenesulfonate; metal salts such as sodium salt, potassium salt and calcium salt; ammonium Examples thereof include, but are not limited to, salts; organic ammonium salts such as methylammonium salts; and amino acid salts such as glycine salts. The same applies to the following antifungal agents and bone disease therapeutic agents.
- anticancer agent refers to those used for the purpose of killing cancer cells, inhibiting cancer cell growth, preventing cancer metastasis, preventing cancer recurrence or preventing cancer occurrence.
- cancer to be administered include malignant melanoma, malignant lymphoma, digestive organ cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract Cancer, breast cancer, liver cancer, pancreatic cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, thyroid cancer, brain tumor, Kaposi Sarcoma, hemangioma, leukemia, polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, myoma, skin cancer, basal cell carcinoma, skin appendage cancer, skin metastasis cancer, Although skin melanoma etc. are mentioned, it is not limited to these.
- the dose when the above compound is administered to a patient can be appropriately set according to the age, weight, type and progression of cancer, symptoms, etc. of the patient.
- 0.1 to 1000 mg / kg body weight, particularly 1 to 100 mg / kg body weight can be administered in 1 to several divided doses.
- the administration route is not particularly limited, and can be administered, for example, by oral administration or parenteral administration such as injection.
- intravenous injection, arterial injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular administration, and the like can be performed.
- the anticancer agent of the present invention can contain a carrier usually used in a pharmaceutical composition in addition to the compound represented by the formula (III) or (IV) or a salt thereof contained as an active ingredient.
- the preparation form can be appropriately selected depending on the purpose of use and the use target, and includes, for example, injections (solutions, suspensions, etc.), tablets, pills, powders, solutions, suspensions, emulsions, granules. It can be used in the form of capsules.
- the carrier include excipients, binders, disintegrants, emulsifiers, solubilizers, dispersants, lubricants, coating agents, colorants, stabilizers, isotonic agents, and the like. It is not limited.
- glazing agents sugars such as lactose, sucrose and glucose, inorganic substances such as starch, calcium carbonate and calcium sulfate, crystalline cellulose, distilled water, purified water, sesame oil, soybean oil, corn oil, olive oil and cottonseed oil What is being done can be illustrated.
- the drug of the present invention can be formulated by a conventional method using these additives.
- the anticancer agent of the present invention can be used in combination with other pharmaceuticals (for example, other anticancer agents).
- the present invention provides a method for treating cancer comprising the step of administering a therapeutically effective amount of a compound represented by formula (III) or (IV) or a pharmaceutically acceptable salt thereof to mammals including humans. Is done.
- the antifungal agent of the present invention contains a compound represented by the following general formula (III) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 5 , R 6 , R 7 , R 8 and R 9 represent alkyl having 1 to 6 carbon atoms, and R 10 represents a hydrogen atom.
- R 5 , R 7 , R 8 and R 9 are preferably alkyl having 1 to 3 carbon atoms, and more preferably methyl.
- R 6 is preferably alkyl having 3 to 6 carbon atoms, more preferably butyl.
- An antifungal agent broadly means a drug having a bactericidal action or a growth inhibitory action against fungi.
- fungi include yeasts, mushrooms, and so-called filamentous fungi (molds), and in particular, fungi such as Candidabalbicans and Candida pseudotropicalis that cause endogenous infections.
- filamentous fungi molds
- the antifungal agent of the present invention is used for treatment of local fungal infection, mucosal infection, systemic fungal infection, etc. caused by, for example, Candida, Trichophyton spp. Or Aspergillus spp. Can do.
- local fungal infection comprising the step of administering a therapeutically effective amount of a compound represented by formula (III) or a pharmaceutically acceptable salt thereof to mammals including humans
- a method of treatment is provided by the present invention.
- the method for measuring the antifungal activity is not particularly limited. For example, using any one or more of the above-mentioned bacteria as a test bacterium, the growth inhibition circle formation in the plate medium is used as an index, or the turbidity of the medium is measured after incubation in a liquid medium for an appropriate period. The antifungal activity can be measured.
- the compound represented by the general formula (III) or a pharmaceutically acceptable salt thereof used as an active ingredient is an antifungal as it is or in combination with various carriers usually used in pharmaceutical compositions as described above. It can be used as an agent.
- the preparation form can be appropriately selected depending on the purpose of use and the intended use. For example, external preparations for skin such as cream or ointment, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules It can be used in the form of suppositories, suppositories, injections (solutions, suspensions, etc.).
- the antifungal agent of the present invention can be administered to mammals including humans.
- the route of administration may be oral or parenteral.
- the dose of the antifungal agent of the present invention should be appropriately increased or decreased according to the patient's age, sex, weight, symptoms, administration route, and other conditions.
- the range is about 1 ⁇ g / kg to 1000 mg / kg, preferably about 10 ⁇ g / kg to 100 mg / kg.
- the above dose may be administered once a day or divided into several times a day.
- the administration period and the administration interval are not particularly limited, and may be administered every day or at intervals of several days.
- the antifungal agent of the present invention is not only used as a pharmaceutical product, but is also taken into the body of humans or animals, such as food, feed, cosmetics, etc. Or it can be used in any product that it is desired to suppress. Moreover, the antifungal agent of this invention can also be used for the surface treatment of a product or a raw material.
- the bone disease treatment agent of the present invention contains a compound represented by the following general formula (IV) or a pharmaceutically acceptable salt thereof as an active ingredient.
- R 11 , R 12 , R 13 , R 14 and R 15 are alkyl having 1 to 6 carbon atoms.
- R 11 , R 13 , R 14 and R 15 are preferably alkyl having 1 to 3 carbon atoms, more preferably methyl.
- R 12 is preferably alkyl having 3 to 6 carbon atoms, and more preferably butyl.
- Bone diseases include both endogenous bone diseases such as bone loss and exogenous bone diseases such as physical fractures, and the agent of the present invention treats the bone disease or shortens the treatment period of the bone disease. Can be used for. Endogenous bone diseases include all diseases involving excessive formation of osteoclasts and / or excessive function in vivo.
- bone diseases include osteoporosis, bone disease-related hypercalcemia, Paget's disease, osteoclastoma, osteosarcoma, arthropathy, rheumatoid arthritis, osteoarthritis, primary hyperthyroidism , Osteopenia, osteoporosis, osteomalacia, traumatic fractures, fatigue fractures, or weakening of bone tissue and fractures due to other diseases such as nutritional disorders and malignant tumors. I don't mean.
- the administration method, dosage form, and dosage of the therapeutic agent for bone disease of the present invention can be appropriately determined according to the usual pharmaceutical method according to the purpose of use.
- it when it is administered to mammals such as humans for the purpose of treatment, it is orally used as powders, granules, tablets, capsules, pills, solvents, etc., or injections, suppositories, transdermal absorption agents, inhalants Etc. can be administered parenterally.
- a therapeutically effective amount of the compound represented by the general formula (IV) or a pharmaceutically acceptable salt thereof is combined with various carriers usually used in the pharmaceutical composition as described above to form a pharmaceutical composition.
- the dose varies depending on the disease state, administration route, patient age, or body weight, but when administered orally to an adult as an active ingredient amount, it is usually 20 to 500 mg / kg / day, preferably 50 to 300 mg / day.
- the dose is usually 10 to 300 mg / kg / day, preferably 20 to 200 mg / kg / day. This may be administered once or divided into several times.
- the present invention provides a method for treating a bone disease, which comprises the step of administering a therapeutically effective amount of a compound represented by formula (IV) or a pharmaceutically acceptable salt thereof to mammals including humans.
- RM-A high production medium (2% potato dextrose (Difco), 1% malt extract (Difco), 1% dry yeast (Asahi beer), 5% Tomato juice, 0.1% K 2 HPO 4 , 0.1% NaCl, 0.03% MgSO 4 ⁇ 7H 2 O, 0.01% NaNO 3 , 0.005% ZnSO 4 ⁇ 7H 2 O, and 0.005% CuSO 4 ⁇ 5H 2 O, pH before autoclave 6.5) or RM-A low production medium (SK2) (1.4% soluble starch, 0.35% glucose, 0.35% yeast extract, 0.21% bactopeptone, 0.21% beef extract (difco), 0.014% KH 2 PO 4 , 0.042 % MgSO 4 , pH 7.6) and cultured at 28 ° C. for 5 days.
- RM-PM 2% potato dextrose (Difco), 1% malt extract (Difco), 1% dry yeast (Asahi beer), 5% Tomato juice, 0.1%
- Sequence analysis was performed by whole genome shotgun sequencing of a reveromycin-producing bacterium. For this purpose, a plasmid library with an insert DNA size of 2-5 kb was prepared. Furthermore, a fosmid library having a 40 kb insert DNA was prepared to cover regions that were difficult to sequence (EPICENTRE Biotechnology). Sequence analysis was performed with 3730xl capillary sequencers (Applied Biosystems) using BigDye terminator ver3.1 kit (Applied Biosystems).
- PCR reaction was performed at 94 ° C. for 2 minutes, 30, 35 or 40 cycles using TaKaRa LA-taq (Takara Bio) at 94 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 30 seconds.
- TaKaRa LA-taq Takara Bio
- KS-F1 ketosynthase
- AT Acyltransferase
- ER enoylreductase
- KS-F1 TSGCSATGGACCCSCAGCAG
- SEQ ID NO: 47 KSAT-F1: GTCGACACSGCCTGYTCSTC
- SEQ ID NO: 49 KSAT-R1: GCGGCGATCTCGCCCTGSGAGTG
- SEQ ID NO: 50 ER-F1: GTGGGCSTGAACTTCCGCGACGT
- ER-F2 GACGTGSTGAMCGSCCTCGGGATG
- ER-F3 GCSGGSGTCGTCACCGCCGTCGG
- ER-R1 CGGCAGCAACCGCAGCGASGCGTC
- ER-R2 GAAAAACCGCAGCGASGCGTC
- the gene fragment specifically amplified in RM-PM medium was ligated to pGEM-T Easy vector (Promega) and transformed into E.coli DH5 ⁇ .
- the plasmid was recovered and sequenced using M13 forward and M13 reverse primers. Analyzed.
- Table 1 shows the ORF size of each identified gene, the estimated function of each ORF, a highly homologous protein,% identity with it, and the access number of the highly homologous protein. All of the ORFs encoded by these genes had low homology with known amino acid sequences.
- revC and revD double-breaking plasmid In order to construct a revC and revD double-breaking plasmid, a 2.5-kb revC fragment and a 2.7-kb revD fragment are used, using fosmid clone (11A02 for revC, 30B04 for revD) as a template, and revC- Eco-Bam-F (5'-CCGGAATTCGGATCCGCCGGCTGCACGAGGAGTCGTCG-3 'SEQ ID NO: 57) and revC-Hind-Asc-R (5'-CCCAAGCTTGGCGCGCCTCGGGTGCGTCCTGCGCGGTG-3' SEQ ID NO: 58), revD is revD-Hind-Asc-F (5 PCR was performed using '-CCCAAGCTTGGCGCGCCTGCCCGACGTGATCGACGACGC-3' SEQ ID NO: 59) and revD-Xho-Bam-R (5'-CCGCTCGAGGGATCCAGCCCCTCGGCCACCGACA
- the amplified revC fragment was digested with EcoR I and HindIII and incorporated into a pET-Duet vector (Novagen) to obtain pET-C.
- the amplified revD fragment was digested with HindIII and XhoI and incorporated into pET-C to obtain pET-CD.
- aphII gene amplified with HindIII site primers (aph-Hind-F: 5'-CTCGAGAAGCTTCAGTGAGTTCGAGCGACTCGAGA-3 'and aph-Hind-R: 5'-CTCGAGAAGCTTCTGGTACCGAGCGAACGCGTA-3' SEQ ID NO: 61, 62) was digested with HindIII and pET -PET-CaphD was constructed by incorporating it into the HindIII site of CD. The obtained pET-CaphD was digested with BamHI, and the resulting revC and revD cassette for double disruption was introduced into the BamHI site of the conjugated plasmid pIM to obtain pIM-CaphD.
- RevL gene disruption was performed by the following method. First, RevL-Hind-F (5'-CCCAAGCTTGGACTTCGCCTGCGCGTTGAACTT-3 'SEQ ID NO: 83) and revL-Hind-R PCR was performed using (5′-CCCAAGCTTAGGCTTCCTGGAGGAAGTCCGTCA-3 ′ SEQ ID NO: 84) as a primer. This gene fragment was introduced into the HindIII site of pUC19.
- revL-Xho-F (5'-CCGCTCGAGAACGCCCCGGAGGGCATCTACTGA-3 'SEQ ID NO: 85) and revL-Xho-R (5'- CCGCTCGAGCTGGTCGACCAGAGCCAGTGATTC-3' SEQ ID NO: 86) primers were used to remove the revL region.
- PCR was performed, and self-ligation was performed after DpnI treatment and XhoI digestion.
- the gene fragment having the RevL-disrupted region was cleaved with HindIII and introduced into the HindIII site of the pIM disruption vector.
- the underlined portion is a homologous sequence used for homologous recombination.
- the underlined part is a restriction enzyme recognition sequence.
- a DNA linking the revG gene (BamHI / HindIII fragment) to the aphII promoter (EcoRI, BamHI fragment) is prepared, and pTYM19 has the function of incorporating DNA into the chromosome. It was introduced into a vector (J Antibiot (Tokyo) 56, 950-6 (2003)). Specifically, a fragment containing the aphII promoter was amplified from Tn5 using the primers of SEQ ID NOs: 75 and 76 below, and incorporated into pTYM19.
- the obtained pTYM19-aphII was digested with BamHI and HindIII, and a revG fragment amplified from 11A02 with the primers of SEQ ID NOs: 77 and 78 shown below was incorporated therein to construct a vector for complementing revG.
- the underlined part is a restriction enzyme recognition sequence.
- a pTYM19 vector J Antibiot (Tokyo) 56, 950-6 (2003) that has a function of integrating a revL gene (BamHI, HindIII fragment) and an aphII promoter (EcoRI, BamHI fragment) into a chromosome. Introduced.
- pTYM19-aphII was digested with BamHI and HindIII, and the following SEQ ID NO: 93 (RevL-Bam-F: CGC GGATCC ATGAACGAATCACTGGCTCTGGTC) and 94 (RevL-Hind-R: CCC AAGCTT TCAGTAGATGCCCTCCGGGGCGTT) primers were amplified from LA fragment rev. Incorporation, a revL complementation vector was constructed.
- pTYM19 was digested with EcoRI and HindIII, and amplified with 11A02 as a template using the following SEQ ID NO: 95 (RevK-Eco-F: CCG GAATTC CACCGGGATGGTGACCTCCAC) and 96 (RevN-Hind-R: CCC AAGCTT TCACGTGTGTTGCGTCCAGGCTTC) primers RevK and revM genes having a raw promoter were incorporated to construct a vector for complementing revK and revM.
- pTYM19-aphII was digested with BamHI and HindIII, and the following SEQ ID NOs: 97 (RevE-Bam-F: CGC GGATCC ATGGACATCACCGCAGCAGTGATC) and 98 (RevE-Hind- (R: CCC AAGCTT TCACCGGTGCGTGAGCACCACCTT)
- the revE fragment amplified from 11A02 with a primer was incorporated to construct a vector for complementing revE.
- E. coli uses GM2929 hsdS :: Tn10 (pUB307 :: Tn7) (Proc Natl Acad Sci USA 107, 2646 (Feb 9, 2010)), and the conjugative transfer vector is pKU250 (Proc Natl Acad Sci USA 107, 2646 (Feb 9, 2010)) was used to optimize the pIM vector by removing the BamHI-KpnI region. For selection of E.
- kanamycin 25 ⁇ g ml -1
- chloramphenicol 30 ⁇ g ml -1
- streptomycin 50 ⁇ g ml -1
- spectinomycin 100 ⁇ g ml -1
- ampicillin 50 ⁇ g ml ⁇ 1
- SY medium 28 °C for 4 hours
- coli GM2929 hsdS Tn10 (pUB307 :: Tn7) containing gene disruption vector at a ratio of 50: 1, MS2 ( 3% soybean meal, 2% D (-) - Man'nittoru, was inoculated into 25 mM MgCl 2, 2% agar) (20 ml) plates. After culturing at 28 ° C. for 20 hours, 20 ⁇ g ml ⁇ 1 thiostrepton and 5 ⁇ g ml ⁇ 1 carmonam were added at final concentrations, and the culturing was further continued for one week. In the case of gene complementation, transformation is completed by selecting a thiostrepton resistant clone.
- ESI-MS analysis a WatersAlliance HPLC system equipped with a mass spectrometer (Q-TRAP, Applied Biosystems) was used. HPLC was analyzed using a solvent A: 0.05% formic acid aqueous solution, solvent B: acetonitrile, XTerra (trademark) MSC18 5 ⁇ m (2.1 mm id ⁇ 150 mm) column at a flow rate of 0.2 ml min ⁇ 1 . After loading the sample onto the column equilibrated with 30% B solvent, linear gradient elution was performed from 30% to 100% B solvent in 20 minutes, and further elution was carried out in 100% B solvent for 20 minutes. Mass spectra were analyzed in ESI-negative mode.
- solvent A 0.05% formic acid aqueous solution
- solvent B acetonitrile
- XTerra (trademark) MSC18 5 ⁇ m (2.1 mm id ⁇ 150 mm) column at a flow rate of 0.2 ml min
- RM-A1a was purified using C 18 -HPLC (Pegasil ODS 10 mm id ⁇ 250 mm) with acetonitrile / 0.05% formic acid aqueous solution (65:35).
- RM-A1e was obtained by culturing the ⁇ revG strain for 5 days or longer.
- RM-A1e was purified using C 18 -HPLC (Pegasil ODS 10 mm id ⁇ 250 mm) with acetonitrile / 0.05% aqueous formic acid (70:30).
- a strain in which ⁇ revG was complemented with the revG gene was cultured in 70 ml of SY medium at 28 ° C. for 2 days (rotary shaker, 150 rpm). 1 ml of the whole culture was added to 70 ml of RM-PM medium and cultured for 5 days. A total of 5 l of culture broth was extracted to obtain 2.5 g of crude extract. This was subjected to SiO 2 column chromatography and eluted with a stepwise gradient of chloroform / methanol. Nine fractions were obtained (9 steps from methanol to 0 to 100%).
- the ⁇ revH ⁇ revI double gene disruption strain showed phenotypes that accumulated RM-A1a and RM-A2a as main intermediate products, and at the same time, trace amounts of RM-A3a and RM-A3b.
- revI reintroduced strain (revH disruption strain) showed the same phenotype as ⁇ revH ⁇ revI double gene disruption strain, but revH reintroduction strain (revI disruption strain) accumulated RM-T. did.
- the RevI gene product acts as RM-T hydroxylase, and the revI disrupted strain is useful as an RM-T producing strain that is a stable RM derivative.
- RevS protein When the function was estimated by heterologous expression and purification of RevS protein using E. coli, it was found to be a CoA ligase specific for unsaturated fatty acids. Heterologous expression and purification of RevT protein using Streptomyces lividans TK23 and estimation of biochemical functions revealed that trans-2-hexenoyl-CoA and trans-2-octenoyl CoA were used as substrates and butylmalonyl CoA and It was found to produce hexylmalonyl CoA. RevN was found to be an esterase that cleaves the ester bond produced by RevH's Bayer-bilger oxidase by expressing and purifying RevH and RevN proteins in E. coli.
- RevM was suggested to be an enzyme that produces RM-A by reducing RM-H using NAD (P) H as a coenzyme. From the above gene disruption analysis, revK and revL were found to be novel enzymes involved in fumarate transfer to RM-T1.
- the gene transfer method was used for gene transfer to enhance production of RMs.
- the promoter of the conjugative transfer vector pTYM19 was replaced with the aphII gene, and a gene for expression downstream was incorporated.
- E. coli uses GM2929 hsdS :: Tn10 (pUB307 :: Tn7), and for selection, ampicillin (50 ⁇ g ml -1 ), chloramphenicol (30 ⁇ g ml -1 ), streptomycin (50 ⁇ g ml -1 ) And spectinomycin (100 ⁇ g ml ⁇ 1 ) was used.
- RMs (RM-A) produced by S. sp. SN-593 strain is about 12 mg L -1 in the SY medium, but the production of RMs in the high production medium is 150-200 mg / L (FIG. 5C).
- the revQ gene disruption lost the ability to produce RMs (FIG. 5D)
- complementing the disrupted strain with the revQ gene restored the production of RMs to the wild strain level (FIG. 5E).
- RevQ is important for transcriptional control.
- the production of RMs increased to a production of about 1 g L ⁇ 1 (FIG. 5F).
- E. coli BL21 Star TM PET28b (+)-revG was introduced into (DE3).
- TB medium containing kanamycin (50 ⁇ g ml ⁇ 1 ) the cells were cultured at 28 ° C. (200 ml) until the OD 600 reached 0.5, and 0.5 mM IPTG was added to induce gene expression. After culturing for 7 hours at 28 ° C., E. coli was recovered by centrifugation.
- the suspension was suspended in 20 ml buffer A (100 mM NaH 2 PO 4 (pH 7.8), 500 mM NaCl, 5 mM imidazole, 10% glycerol) containing 0.5 mg lysozyme ml ⁇ 1 and 125 U benzonase and subjected to ultrasonic disruption. After centrifugation, the supernatant was applied to a Ni-NTA (2 ⁇ 2 cm) (Qiagen) column.
- buffer A 100 mM NaH 2 PO 4 (pH 7.8), 500 mM NaCl, 5 mM imidazole, 10% glycerol
- RM-A2a was produced in 1 minute of reaction
- RM-A3a and RM-A3b were produced in 10 minutes of reaction. From this, RM-A3a having a spiroketal ring under acidic HPLC conditions from RM-A1a in a non-cyclized state via reaction intermediate RM-A2a and further unstable intermediate C15-dehydro-RM-A2a and It was found to be converted to RM-A3b.
- the NMR charts for RM-A1a, RM-T, RM-A2a, RM-A3a and A3b are shown in FIGS.
- RevJ mass expression and functional analysis (1) Preparation of E. coli heterologous expression vector of revJ gene Using template fosmid 11A02 containing revJ gene, the following primers, and PrimeSTAR HS DNA polymerase (TaKaRa), 98 ° C for 10 seconds, 98 ° C for 10 seconds, 62 ° C for 5 seconds, A 25-cycle PCR reaction was performed at 68 ° C for 1.5 minutes.
- revJ gene fragment was cleaved with restriction enzymes (NdeI and XhoI) and then introduced into pET28b (+) b (polyhistidine fusion protein expression vector: Novagen) used for E. coli heterologous expression to produce pET28b (+)-revJ. .
- the suspension was suspended in 20 ml buffer A (100 mM NaH 2 PO 4 (pH 7.8), 500 mM NaCl, 5 mM imidazole, 10% glycerol) containing 0.5 mg lysozyme ml ⁇ 1 and 125 U benzonase and subjected to ultrasonic disruption. After centrifugation, the supernatant was applied to a Ni-NTA (2 ⁇ 2 cm) (Qiagen) column.
- buffer A 100 mM NaH 2 PO 4 (pH 7.8), 500 mM NaCl, 5 mM imidazole, 10% glycerol
- HL-60 cells human acute promyelocytic leukemia cell line
- K562 cells human chronic myeloid leukemia cell line
- Fetal Bovine Serum Fetal Bovine Serum (Nichirei In a medium supplemented with 0.5% penicilin / streptomycin solution (manufactured by Invitrogen) and maintained in a 37 ° C., 5% CO 2 humidity culture.
- tsFT210 cells (mouse breast cancer cell CDC2 temperature-sensitive strain) were cultured in RPMI1640 medium (Invitrogen) with 5% calf serum (Hyclone) and 0.5% penicilin / streptomycin solution in 32 ° C, 5% CO 2 humidity. Maintained in medium culture. HL-60 cells and K562 cells were seeded in a 96-well plate (manufactured by IWAKI) at 1.5 ⁇ 10 4 cells / well / 100 ⁇ l. tsFT210 cells were seeded in a 96-well plate at 1.6 ⁇ 10 4 cells / well / 100 ⁇ l.
- HL-60 cells and K562 cells were cultured in a humidity of 37 ° C and 5% CO 2
- tsFT210 cells were humidified at 32 ° C and 5% CO 2 .
- WST-8 Reagent Viable Cell Count Reagent SF Solution
- the tsFT210 cells were maintained for 1 hour in a culture at 32 ° C. and 5% CO 2 in a medium culture for 30 minutes. After the reaction, the absorbance at 450 nm was measured using a microplate reader (manufactured by PerkinElmer), and the cell proliferation rate was determined from the measured values.
- Bone marrow cells were collected from the femur and tibia of 5-week-old male ddY mice (Japan SLC), and 10% fetal bovine serum, 0.5% in ⁇ -MEM medium (Sigma-Aldrich) Type I collagen-coated plate (manufactured by IWAKI) in a medium supplemented with% penicilin / streptomycin solution, 50 ng / ml human M-CSF (Leukoprol, manufactured by Kyowa Hakko) and 1 ng / ml human TGF- ⁇ 1 (manufactured by R & D Systems) ) And maintained for 3 days in a 37 ° C., 5% CO 2 humidified culture.
- ⁇ -MEM medium Sigma-Aldrich
- Bone marrow macrophage cells in medium supplemented with 10% fetal-bovine serum, 0.5% penicilin / streptomycin solution, 50 ng / ml human M-CSF, 50 ng / ml human soluble RANKL (Peprotech) The cells were maintained for 3 days in a humidity culture at 37 ° C. and 5% CO 2 to differentiate into osteoclasts.
- Each of the drugs shown in Table 5 was added to osteoclasts at 0.5% (v / v) and maintained for 24 hours in a 37 ° C., 5% CO 2 humidity culture.
- the cells are reacted with PBS solution containing 3.7% formalin for 30 minutes at room temperature. After removing the solution, further reacted with acetone / ethanol solution (1: 1 vol / vol) for 1 minute at room temperature to remove the solution. Dried. React the immobilized cells with TRAP solution [50 mM sodium tartrate, 90 mM sodium acetate, 0.01% naphthol AS-MX phosphate (Sigma), 0.05% fast red violet LB salt (Sigma), pH 5.0] at room temperature for 30 minutes. And then washed with distilled water. The number of TRAP-positive multinucleated osteoclasts was counted to determine the survival rate.
- TRAP solution 50 mM sodium tartrate, 90 mM sodium acetate, 0.01% naphthol AS-MX phosphate (Sigma), 0.05% fast red violet LB salt (Sigma), pH 5.0
- RM-E Wild strain Streptomyces sp. SN-593
- SY medium 1 ml of the preculture was inoculated into RM-PM (70 ml) medium, and further cultured for 5 days .
- an equal amount of acetone was added to a total of 3 L of the culture solution and stirred, then the acetone was removed, the pH was adjusted to 4 with acetic acid, and an equal amount of ethyl acetate was added to perform extraction twice.
- RM-A2b After culturing a wild strain (Streptomyces sp. SN-593) in SY medium for 2 days, inoculate 200 ml of the precultured medium in RM-PM (14 L) medium and culturing for 4 days in a jar fermenter. went. Thereafter, an equal amount of acetone was added and stirred, then the acetone was removed, the pH was adjusted to 4 with acetic acid, and an equal amount of ethyl acetate was added to perform extraction twice. After C 18 -MPLC chromatography, 0.05% formic acid-acetonitrile 55-100% gradient elution was performed using Pegasil ODS to purify 0.33 mg.
- RM-T ethylester and RM-T methylester Wild strains (Streptomyces sp. SN-593) were cultured in SY medium for 2 days. Thereafter, 1 ml of the preculture was inoculated into RM-PM (70 ml) medium and cultured. Three days later, ethanol was added to a final concentration of 1%, and the cells were further cultured for 2 days. Then, after adding an equal amount of acetone to a total of 1.4 L of the culture solution and stirring, the acetone was removed, the pH was adjusted to 4 with acetic acid, and the extraction was performed twice by adding an equal amount of ethyl acetate.
- RM-A8a was prepared with the following reaction solution composition.
- Solution (30 ml) containing 50 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 1 mM NAD + , 0.7 mM NADPH, 0.1 mM FAD, 0.04 mM RM-A1a, 100 nmol purified RevG and 31 nmol purified RevH
- the reaction was carried out at 30 ° C for 150 minutes. Next, 20 nmol of purified RevJ was added, and the reaction was further carried out at 30 ° C. for 120 minutes.
- reaction solution was extracted twice with an equal amount of ethyl acetate, dehydrated with sodium sulfate, and then ethyl acetate was distilled off with an evaporator.
- the residue was dissolved in methanol and subjected to HPLC (column: PEGASIL ODS (20 mm ⁇ 250 mm, Senshu Chemical Co., Ltd.)), and the elution was performed with 85% acetonitrile at a flow rate of 8 ml min ⁇ 1. After 25 minutes, acetonitrile / water was distilled off with an evaporator to obtain about 0.3 mg of RM-A8a.
- RM-A6a and RM-A9a are produced by the revE disruption strain (FIG. 14).
- the revE gene-disrupted strain was cultured in SY medium for 2 days, then 1 ml of this preculture was inoculated into RM-PM (70 ml) medium and main culture was performed for 5 days.
- An equal volume of acetone was added to a total of 5 L of the culture solution to extract metabolites, remove acetone, and adjust the pH to 4 with acetic acid.
- an equal amount of ethyl acetate was added and extraction was performed three times. Ethyl acetate was removed to obtain 3 g of a crude fraction.
- the present invention is useful in the field of substance production and medicine.
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Abstract
Description
[1]リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌であって、配列番号36のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードするrevQ遺伝子の発現が親株と比較して増大するように改変され、それにより前記生産能が該親株よりも向上したストレプトマイセス属細菌。
[2]revQ遺伝子のコピー数を高めること、またはrevQ遺伝子のプロモーターの改変によってrevQ遺伝子の発現が増大した、[1]に記載の細菌。
[3]ストレプトマイセス sp. SN-593株をrevQ遺伝子の発現が増大するように改変することによって得られる、[1]または[2]に記載の細菌。
[4] [1]~[3]のいずれかに記載のストレプトマイセス属細菌を培地で培養し、培地中にリベロマイシンAまたはその合成中間体を蓄積させ、その培養物からリベロマイシンAまたはその合成中間体を回収することを含む、リベロマイシンAまたはその合成中間体の製造方法。
[5]配列番号36のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードし、リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌に導入したときに、該生産能を向上させるポリヌクレオチド。
[6]配列番号35の121-951の塩基配列とストリンジェントな条件でハイブリダイズし、リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌に導入したときに、該生産能を向上させるポリヌクレオチド。
[7]配列番号14のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列を有するRevGタンパク質を下記化合物(I)に作用させることにより、該化合物(I)を下記化合物(II)に変換する工程を含む、化合物(II)の製造方法。
[8] 前記RevGタンパク質とともに配列番号20のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列を有するRevJタンパク質を化合物(I)に作用させる、[7]に記載の化合物(II)の製造方法。
[9]化合物(I)が下記化合物(i)であり、化合物(II)が下記化合物(ii)である、[7]または[8]に記載の方法。
[11] [10]に記載のタンパク質をコードするポリヌクレオチド。
[12]配列番号13の121-939の塩基配列の相補鎖とストリンジェントな条件でハイブリダイズする、[11]に記載のポリヌクレオチド。
[13]配列番号2、4、6、8、10、12、16、18、20、22、24、26、28、30、34、38、40、42または44のアミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードし、リベロマイシン生合成関連タンパク質をコードするポリヌクレオチド。
[14]下記一般式(III)または(IV)で表される化合物又はその薬学的に許容される塩を有効成分とする、抗癌剤。
[15]下記一般式(III)で表される化合物又はその薬学的に許容される塩を有効成分とする、抗真菌剤。
R5、R6、R7、R8およびR9は炭素数1~6のアルキルを示し、R10は水素原子を示す。
[16]下記一般式(IV)で表される化合物又はその薬学的に許容される塩を有効成分とする、骨疾患治療剤。
R11、R12、R13、R14およびR15は炭素数1~6のアルキルを示す。
<1>本発明の細菌およびそれを用いたリベロマイシンAまたはその合成中間体の製造法
本発明の細菌は、リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌であって、配列番号36のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードするrevQ遺伝子の発現が親株と比較して増大するように改変され、それにより前記生産能が該親株よりも向上したストレプトマイセス属細菌である。
さらに、revQ遺伝子は、配列番号36のアミノ酸配列において、1または数個のアミノ酸が置換、欠失、挿入または付加された配列を有するアミノ酸配列をコードし、リベロマイシンAまたはその合成中間体の生産能を向上させる遺伝子であってもよい。ここで、1または数個とは、好ましくは、1~20個、より好ましくは1~10個、特に好ましくは1~5個を意味する。
ストレプトマイセス・プルニカラー(Streptomyces prunicolor)
ストレプトマイセス・シナモネウス(Streptomyces cinnamoneus)
ストレプトマイセス・クロモフスカス(Streptomyces chromofuscus)
ストレプトマイセス・リビダンス(Streptomyces lividans)
ストレプトマイセス・アキヨシエンシス(Streptomyces akiyoshiensis)
ストレプトマイセス・アズレウス(Streptomyces azureus)
ストレプトマイセス・ハワイエンシス(Streptomyces hawaiiensis)
ストレプトマイセス・テンダエ(Streptomyces tendae)
ストレプトマイセス・バージニア(Streptomyces virginiae)
ストレプトマイセス・アマクサエンシス(Streptomyces amakusaensis)
ストレプトマイセス・アンチビオティカス(Streptomyces antibioticus)
ストレプトマイセス・チバエンシス(Streptomyces chibaensis)
ストレプトマイセス・アルバス(Streptomyces albus)
ストレプトマイセス・リンコエンシス(Streptomyces lincolnensis)
ストレプトマイセス・カナマイセティカス(Streptomyces kanamyceticus)
ストレプトマイセス・カスガエンシス(Streptomyces kasugaensis)
ストレプトマイセス・セリカラー(Streptomyces coelicolor)
ストレプトマイセス・グリセウス(Streptomyces griseus)
ストレプトマイセス・エバミチリス(Streptomyces avermitilis)
ストレプトマイセス・アンボファシエンス(Streptomyces ambofaciens)
ストレプトマイセス・フラディア(Streptomyces fradiae)
revQ遺伝子を組み込むことができるプラスミドベクターとしては、宿主細菌内での複製増殖機能を司る遺伝子を少なくとも含むものであれば特に制限されない。上記自立複製に関与するベクターとして、例えば、プラスミド DNA、ウイルス、バクテリオファージ、細菌の染色体等が挙げられる。
pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986)、
pKC1064(Gene 103,97-99 (1991))、
pUWL-KS (Gene 165,149-150 (1995))、
pIJ702(J. Gen. Microbiol. 129:2703-2714(1983))、
pIJ8600(Microbiology 145:2221-2227(1999))
ファージDNAとしてはλファージ(Charon4A、Charon21A、EMBL3、EMBL4、λgt10、λgt11)等が挙げられる。
また、DNAが導入されたことの確認は、選択マーカー遺伝子(例えばアンピシリン耐性遺伝子、テトラサイクリン耐性遺伝子、クロラムフェニコール耐性遺伝子、カナマイシン耐性遺伝子等)を用いて行うとよい。
また、revQ遺伝子の発現増強は、宿主染色体上でrevQ遺伝子のプロモーターを置換または改変することによっても行うことができる。プロモーター置換の方法としては、例えば、公知の相同組み換え法やsacB遺伝子を用いる方法(Schafer,A.et al.Gene 145 (1994)69-73)が挙げられる。
抗生物質のチオストレプトン添加により転写が誘導されるtipAプロモーター(Gene 94:53-59、1990;Gene 103: 97-99、1991;Gene 166: 133-137、1995)、グリセロールにより誘導されるglyCABプロモーター(Mol.Microbiol. 12: 737-745、1994)およびマイトマイシンCにより誘導されるmcrABプロモーター(Gene 175: 261-267、1996)が発現ベクターに利用されている。
また、大腸菌で用いられるlacプロモーターやtacプロモーターやtrcプロモーターなどが挙げられる。
本発明はまた、配列番号14のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列を有するRevGタンパク質を下記化合物(I)に作用させることにより、該化合物(I)を下記化合物(II)に変換する工程を含む、化合物(II)の製造方法を提供する。
なお、RevGタンパク質とともにRevJタンパク質を化合物(I)に作用させてもよい。
さらに、RevGタンパク質は、配列番号16のアミノ酸配列において、1または数個のアミノ酸が置換、欠失、挿入または付加された配列を有し、化合物(I)を化合物(II)に変換する反応を触媒する活性を有するタンパク質であってもよい。ここで、1または数個とは、好ましくは、1~20個、より好ましくは1~10個、特に好ましくは1~5個を意味する。
RevJタンパク質としては、配列番号20のアミノ酸配列を有するストレプトマイセスsp. SN-593株由来のタンパク質が挙げられるが、配列番号20のアミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上、特に好ましくは99%以上の同一性を有し、RevGとともに化合物(I)を化合物(II)に変換する反応を触媒する活性を有するタンパク質であってもよい。
さらに、RevJタンパク質は、配列番号20のアミノ酸配列において、1または数個のアミノ酸が置換、欠失、挿入または付加された配列を有し、RevGとともに化合物(I)を化合物(II)に変換する反応を触媒する活性を有するタンパク質であってもよい。ここで、1または数個とは、上記と同様である。
RevGタンパク質およびRevJタンパク質はストレプトマイセスsp. SN-593株などのストレプトマイセス属細菌から精製することによって得ることもできるが、revG遺伝子およびRevJタンパク質を適当な宿主(ストレプトマイセス属細菌、エシェリヒア属細菌、酵母など)または無細胞系で発現させた後に、RevGタンパク質およびRevJタンパク質を精製することによって得ることもできる。その際に、精製の簡便のため、ポリヒスチジンやGSTなどのタグを融合させて発現させ、そのタグに対するアフィニティを利用して精製することが好ましい。
本発明は、上記revG遺伝子およびrevQ遺伝子に加えて、さらに、下記の各新規ポリヌクレオチドを提供する。
revC遺伝子:配列番号1(コードされるアミノ酸配列は配列番号2)
revA遺伝子:配列番号3(コードされるアミノ酸配列は配列番号4)
revB遺伝子:配列番号5(コードされるアミノ酸配列は配列番号6)
revD遺伝子:配列番号7(コードされるアミノ酸配列は配列番号8)
revE遺伝子:配列番号9の121-1221(コードされるアミノ酸配列は配列番号10)
revF遺伝子:配列番号11の121-1569(コードされるアミノ酸配列は配列番号12)
revH遺伝子:配列番号15の121-1641(コードされるアミノ酸配列は配列番号16)
revI遺伝子:配列番号17の121-1311(コードされるアミノ酸配列は配列番号18)
revJ遺伝子:配列番号19の121-1128(コードされるアミノ酸配列は配列番号20)
revK遺伝子:配列番号21の121-1050(コードされるアミノ酸配列は配列番号22)
revL遺伝子:配列番号23の121-1191(コードされるアミノ酸配列は配列番号24)
revM遺伝子:配列番号25の121-1080(コードされるアミノ酸配列は配列番号26)
revN遺伝子:配列番号27の121-1035(コードされるアミノ酸配列は配列番号28)
revO遺伝子:配列番号29の121-777(コードされるアミノ酸配列は配列番号30)
revP遺伝子:配列番号33の121-792(コードされるアミノ酸配列は配列番号34)
revR遺伝子:配列番号37の121-1119(コードされるアミノ酸配列は配列番号38)
revS遺伝子:配列番号39の121-1857(コードされるアミノ酸配列は配列番号40)
revT遺伝子:配列番号41の121-1449(コードされるアミノ酸配列は配列番号42)
revU遺伝子:配列番号43の121-2889(コードされるアミノ酸配列は配列番号44)
本発明の抗癌剤は下記一般式(III)または(IV)で表される化合物又はその薬学的に許容される塩を有効成分として含む。
R5、R6、R7、R8およびR9は炭素数1~6のアルキルを示し、R5、R7、R8およびR9は好ましくは炭素数1~3のアルキルであり、より好ましくはメチルである。R6は好ましくは炭素数3~6のアルキルであり、より好ましくはブチルである。
R10は水素原子または炭素数1~5のアルキルを示し、好ましくは水素原子、メチルまたはエチルである。
R11、R12、R13、R14およびR15は炭素数1~6のアルキルを示し、R11、R13、R14およびR15は好ましくは炭素数1~3のアルキルであり、より好ましくはメチルである。R12は好ましくは炭素数3~6のアルキルであり、より好ましくはブチルである。
本発明の抗真菌剤は、下記一般式(III)で表される化合物又はその薬学的に許容される塩を有効成分として含む。
R5、R6、R7、R8およびR9は炭素数1~6のアルキルを示し、R10は水素原子を示す。R5、R7、R8およびR9は好ましくは炭素数1~3のアルキルであり、より好ましくはメチルである。R6は好ましくは炭素数3~6のアルキルであり、より好ましくはブチルである。
本発明の骨疾患治療剤は、下記一般式(IV)で表される化合物又はその薬学的に許容される塩を有効成分として含む。
R11、R12、R13、R14およびR15は炭素数1~6のアルキルを示す。R11、R13、R14およびR15は好ましくは炭素数1~3のアルキルであり、より好ましくはメチルである。R12は好ましくは炭素数3~6のアルキルであり、より好ましくはブチルである。
さらに、式(IV)で示される化合物又はその薬学的に許容される塩の治療有効量を、ヒトを含む哺乳類動物に投与する工程を含む、骨疾患の治療方法が本発明により提供される。
以下の手順に従い、リベロマイシン生産菌(S. sp. SN-593)からのリベロマイシン生合成遺伝子クラスターの取得を行った。
リベロマイシン生産菌(S. sp. SN-593) 培養
MS寒天プレート(2 % 大豆粉, 2 % D(-)-マンニトール, 2 % 寒天) で 2週間28℃で培養を行い、胞子を着生させた。
一白金耳の胞子をSY培養液 (0.1 % 酵母抽出物, 0.1 % NZ-アミン, 1 % 可溶性デンプン, pH7.0) 70 ml(500ml K1フラスコ)に植菌し2日間28℃で150 rpmで培養した。この前培養液1 mlを70 mの RM-A高生産培地(RM-PM)(2% ポテトデキストロース(Difco), 1% モルト抽出物(Difco), 1% 乾燥酵母(アサヒビール), 5%トマトジュース, 0.1% K2HPO4, 0.1% NaCl, 0.03% MgSO4・7H2O, 0.01% NaNO3, 0.005% ZnSO4・7H2O, and 0.005% CuSO4・5H2O, オートクレーブ前pH 6.5)又はRM-A低生産培地(SK2) (1.4 %可溶性デンプン, 0.35 %グルコース, 0.35 %酵母抽出物, 0.21 % バクトペプトン, 0.21 % ビーフ抽出液 (difco), 0.014 % KH2PO4, 0.042 % MgSO4, pH 7.6)に植菌して5日間28℃で培養した。
リベロマイシン生産菌の全ゲノムショットガンシークエンスにより配列解析を行った。このためにインサートDNAサイズが2-5 kbのプラスミドライブラリーを作成した。更に配列解析が難しい領域を網羅するために40 kbのインサートDNAを有するフォスミドライブラリーを作成した(EPICENTRE Biotechnology)。配列解析はBigDye terminator ver3.1 kit (Applied Biosystems)を用いて3730xl capillary sequencers (Applied Biosystems)で解析した。
ポリケチド合成酵素(PKS)配列を持つ遺伝子を特異的に増幅するためのプライマーを設計し、これらのプライマーによりリベロマイシン生産培地での培養時に特異的に増幅される(リベロマイシン低生産性培地培養時には増幅されない)遺伝子の取得を試みた。
リベロマイシン生産菌の胞子をSK2 培地(70 ml)中で2日間、28℃で培養後、この前培養液1 mlをそれぞれ70 ml の生産培地(RM-PM)及び低生産性培地(SK2)に植菌した。60時間後、培養液5 mlから全RNAをTRIzol Max Bacterial RNA isolation kit (Invitrogen)を用いて抽出した。DNase I により混在する染色体DNAを除去した後、SuperScript III (Invitrogen)を用いて逆転写反応を行った。
ketosynthase (KS), Acyltransferase (AT), enoylreductase (ER):
KS-F1 :TSGCSATGGACCCSCAGCAG, (配列番号47)
KS-R1: CCSGTRCCGTGCGCCTCSAC, (配列番号48)
KSAT-F1: GTCGACACSGCCTGYTCSTC, (配列番号49)
KSAT-R1: GCGGCGATCTCGCCCTGSGAGTG, (配列番号50)
ER-F1: GTGGGCSTGAACTTCCGCGACGT, (配列番号51)
ER-F2: GACGTGSTGAMCGSCCTCGGGATG, (配列番号52)
ER-F3: GCSGGSGTCGTCACCGCCGTCGG, (配列番号53)
ER-R1: CGGCAGCAACCGCAGCGASGCGTC, (配列番号54)
ER-R2: GGTCTTGCCCATCTCSASGAASCG, (配列番号55)
ER-R3: GACGACCTTGCCCACATGACG. (配列番号56)
KS, KS-AT 及び ERプライマーは、それぞれ0.6, 1.5及び 0.7 kb増幅するようにデザインした。
以上のようにして得られた候補遺伝子クラスターが確かにRM-A生合成遺伝子クラスターであることを確認するために、ポリケチド生合成遺伝子revC, revDの二重遺伝子破壊を行った。revCの3'末端領域及びrevDの5'末端領域を欠失させ、内部にカナマイシン耐性化に関わるaphII遺伝子(約1.6 kb)を組み込んだ。更にaphII遺伝子の上流及び下流に約2.5 kbの相同組換えDNA領域を含む接合伝達ベクターを調製した。同様な手法でrevH, revIの二重遺伝子破壊株作製用のプラスミドを構築した(図4A)。また、クラスター境界のorf(-1)(配列番号31)、orf1(配列番号45)及びrevGの遺伝子破壊を行うために、各遺伝子の内部配列をaphII遺伝子に置き換え、同様に上流及び下流に約2.5 kbの相同組換えDNA領域を含む遺伝子破壊ベクターを調製した。具体的な手順は以下のとおりである。
revE, revK, revMについても同様の方法で遺伝子破壊を行った。revE,revK, revM破壊のスキームを図12に示した。
破壊した遺伝子の機能相補を確認するために、aphIIプロモーター(EcoRI、BamHI断片)にrevG遺伝子(BamHI/HindIII断片)を連結したDNAを作製し、染色体へのDNA組み込み機能を有するpTYM19ベクター(J Antibiot (Tokyo) 56, 950-6 (2003))に導入した。具体的には、aphII プロモーターを含む断片をTn5 から下記配列番号75と76のプライマーを用いて増幅し、pTYM19に組みこんだ。得られたpTYM19-aphII をBamHIとHindIIIで消化し、ここに下記配列番号77と78のプライマーで11A02から増幅したrevG断片を組み込んで、revG 相補用ベクターを構築した。
また、pTYM19をEcoRIとHindIIIで消化し、ここに下記配列番号95(RevK-Eco-F: CCGGAATTCCACCGGGATGGTGACCTCCAC)と96(RevN-Hind-R: CCCAAGCTTTCACGTGTGTTGCGTCCAGGCTTC)プライマーで11A02を鋳型として増幅した内生のプロモーターを有するrevK、revM遺伝子を組み込み、revKおよびrevM相補用ベクターを構築した。
また、破壊したrevE遺伝子の機能相補を確認するために、pTYM19-aphII をBamHIとHindIIIで消化し、ここに下記配列番号97(RevE-Bam-F: CGCGGATCCATGGACATCACCGCAGCAGTGATC)と98(RevE-Hind-R: CCCAAGCTTTCACCGGTGCGTGAGCACCACCTT)プライマーで11A02から増幅したrevE断片を組み込んで、revE相補用ベクターを構築した。
遺伝子導入は大腸菌からリベロマイシン生産菌への接合伝達法により行った。大腸菌はGM2929 hsdS::Tn10 (pUB307::Tn7) (Proc Natl Acad Sci U S A 107, 2646 (Feb 9, 2010))を使用し、接合伝達ベクターは、pKU250 (Proc Natl Acad Sci U S A 107, 2646 (Feb 9, 2010))からBamHI-KpnI 領域を除くことにより最適化を行ったpIMベクターを使用した。遺伝子破壊ベクターを有する大腸菌の選別には、カナマイシン (25 μg ml-1)、クロラムフェニコール (30 μg ml-1)、ストレプトマイシン (50 μg ml-1)及びスペクチノマイシン(100 μg ml-1)を含むLB (1% トリ プトン, 0.5% 酵母抽出物, 1% NaCl)培地を利用した。遺伝子相補ベクターを有する場合には、カナマイシンの代わりにアンピシリン(50 μg ml-1)を用いた。
リベロマイシン生産菌の胞子を調製し、SY培地で28℃、4時間培養後、遺伝子破壊ベクターを含む大腸菌GM2929 hsdS::Tn10 (pUB307::Tn7) と50:1の割合で混合し、MS2 (3 % 大豆粉, 2 % D(-)-マンニットール, 25 mM MgCl2, 2 % 寒天) (20 ml)プレート上に植菌した。28 ℃、20時間培養後、終濃度で20 μg ml-1 チオストレプトン、5 μg ml-1 カルモナムを添加し、更に1週間培養を進めた。遺伝子相補の場合には、チオストレプトン耐性のクローンを選別で形質転換が完了するが、遺伝子破壊の場合には、更に0.4 μg ml-1のリボスタマイシンを含むMSプレートで二次選別を行った後、得られた耐性クローンを薬剤を含まないSY培地(70 ml)で液体培養、MSプレート上で胞子形成を行い、単一の胞子から得られたコロニーを調製し、リボスタマイシン耐性、チオストレプトン感受性のクローンを選別した。
遺伝子破壊株の確認にはサザンハイブリダイゼーション解析を行った(図3b)。PrimeSTAR HS DNA polymerase (TaKaRa)を用いて各プローブ調製を行った。反応は、98℃10 秒, 25 サイクル98℃10秒後, 67℃ 5秒, 68℃ 2.5 分の25サイクルPCR反応、又は98℃ 10秒後, 98℃ 10秒, 67℃ 5秒, 68℃ 40秒の25サイクルPCR反応を行った。増幅したDNA 断片はゲル切り出し精製し、AlkPhos Direct Labelling Reagents (GE Healthcare)により標識を行った。
野生株(Streptomyces sp. SN-593)及び遺伝子破壊株をSY培地で2日間培養を行った。その後、前培養液1 ml をRM-PM (70 ml)培地に植菌し更に5日間培養を行った。その後、等量のアセトンを加え撹拌後、アセトンを除去し、酢酸でpHを4に調整し、等量の酢酸エチルを添加して抽出を2回行った。その後、酢酸エチルを除去し、メタノール20 mlに溶解し、LC-MSにて解析を行った。ESI-MS 解析は、マススペクトロメーター (Q-TRAP, Applied Biosystems)を装備したWatersAlliance HPLCシステムを用いた。HPLCは、A溶媒:0.05% ギ酸水溶液、B溶媒:アセトニトリル、XTerra(商標)MSC18 5 μm (2.1 mm i.d. x 150 mm)カラムを用いて、流速0.2 ml min-1で解析を行った。30%B溶媒で平衡化されたカラムに試料をロード後、20分間で30%から100%B溶媒のリニアグラジエント溶出を行い、さらに20分間100%B溶媒で溶出を行った。マススペクトルはESI-ネガティブモードで解析を行った。
orf(-1)破壊株、およびorf1破壊株を解析した結果、これらの株はいずれもリベロマイシン類の生産において野生株と変わらなかった。したがって、これらの遺伝子はリベロマイシン生合成系に関与していないことが考えられた。
大腸菌を用いて、RevS蛋白質を異種発現し精製することにより、機能を推定したところ、不飽和脂肪酸に特異的なCoAリガーゼであることが判明した。
放線菌(Streptomyces lividans TK23)を用いてRevT蛋白質を異種発現し精製し、生化学的機能を推定したところ、トランス-2-ヘキセノイル-CoAやトランス‐2-オクテノイルCoAを基質として、ブチルマロニルCoAやヘキシルマロニルCoAを生成することが判明した。
大腸菌を用いて、RevH、RevN蛋白質を異種発現し精製することにより、RevNは、RevHのバイアービリガー酸化酵素により生成したエステル結合を切断するエステラーゼであることが判明した。
3級水酸基へのヘミサクシニネートの構築は困難であり、有機化学的には、15,000気圧という超高圧下での反応が行われているが、リベロマイシン生産菌は常温常圧下で生合成反応を行う事が出来る。C18-hydroxy RM-T (RM-T1)からRM-Aを生成するヘミサクシニネート化機構を明らかにするためにrevK, revL, revMの各遺伝子破壊解析を行った。遺伝子破壊とサザンハイブリダイゼーションによる確認結果を図12に示す(ΔrevK (図12 a,d)、ΔrevL (図12 b,e)、ΔrevM (図12 c,f))。次に、revK, revL及びrevM遺伝子破壊株に蓄積する生合成中間産物の解析を行った。
機能未知のrevK遺伝子の破壊株には、主にm/z 559 [M-H]-のC18-hydroxy RM-T (RM-T1)及びRM-T1の5,6-spiroacetal体(RM-T2)が蓄積した(図13c)。また、マイナー産物として、RM-T1とRM-T2の水酸化体と予想されるm/z 575 [M-H]-の2つのピーク(RM-T3, RM-T4)が検出された(図13c)。更に、遺伝子破壊株にrevK遺伝子を再導入すると、RM-A生産が回復することが確認できた(図13d)。
機能未知のrevL遺伝子の破壊株には、revK破壊株と同様に、主にm/z 559 [M-H]-のRM-T1及びRM-T2が蓄積し、マイナー産物として、m/z 575 [M-H]-の2つのピーク(RM-T3, RM-T4)が確認された(図13e)。 更にrevL遺伝子を相補する事によってRM-A生産が回復する事が確認出来た(図13f)。RevK及びRevL破壊株に蓄積する代謝産物が同一であることから、RM-T1以後の反応に二つの酵素が必要であることが示唆された。また、相補株中に、m/z 675 [M-H]-を示す新規類縁体(RM-T5: C14 hydroxy RM-A)が見出された(図13f) 。
機能未知のrevM遺伝子の破壊株は、RM-T1,T2,T3,T4に加え、m/z 657のRM誘導体を生産した(図13g)。また、revM遺伝子を導入する事によってRM-A生産が回復する事を確認出来た(図13h)。RevMは、NAD(P)Hを補酵素としてRM-Hを還元することでRM-Aを生成する酵素であることが示唆された。 以上の遺伝子破壊解析から、revK及びrevLはRM-T1へのフマル酸転移に関わる新規酵素であることが判明した。
revE遺伝子破壊とサザンハイブリダイゼーションによる確認結果を図14a,bに示す。ΔrevE株では、リベロマイシンAは生成せず、主産物としてm/z 529 [M-H]- のRM-A6a, 時間経過的にm/z 645 [M-H]- のRM-A9aが蓄積した(図14d)。そして、ΔrevE株においてrevEを相補すると、リベロマイシンAが生成した(図14e)。したがって、revEはRM-A6aとリベロマイシンAの間の反応に関与することが判明した。
RM類の生産増強のための遺伝子導入には、接合伝達法を行った。構成的発現のために、接合伝達ベクターpTYM19のプロモーターをaphII遺伝子に置き換え、その下流に発現にさせる遺伝子を組み込んだ。大腸菌は、GM2929 hsdS::Tn10 (pUB307::Tn7)を使用し、選別には、アンピシリン (50 μg ml-1)、クロラムフェニコール (30 μg ml-1)、ストレプトマイシン (50 μg ml-1)及びスペクチノマイシン(100 μg ml-1)を含むLB培地を利用した。リベロマイシン生産菌の胞子を調製し、SY培地で28 ℃ 、4時間培養後、接合伝達ベクターを含む大腸菌と50:1の割合で混合し、MS2 (20 ml)プレート上に植菌した。28 ℃、20時間培養後、終濃度でチオストレプトン(20 μg ml-1)、カルモナム(5 μg ml-1)を添加し、1週間培養を行い、チオストレプトン耐性形質転換株を選別した。
野生株(Streptomyces sp. SN-593)、revQ遺伝子破壊株、及び遺伝子導入株をSY培地で2日間培養を行った。その後、前培養液1 ml をRM-PM培地に植菌し更に5日間培養を行った。その後、等量のアセトンを加え撹拌後、アセトンを除去し、酢酸でpHを4に調整し、等量の酢酸エチルを添加して抽出を2回行った。その後、酢酸エチルを除去し、メタノールに溶解し、LC-MS解析を行った。
revG遺伝子の大腸菌異種発現ベクター作製
revG遺伝子を含む鋳型フォスミド11A02、下記のプライマー、およびPrimeSTAR HS DNA polymerase (TaKaRa)を用い、98℃ 10秒後、98℃ 10秒, 62℃ 5秒, 68℃ 1.5分の25サイクルPCR反応を行った。
5'-GGAATTCCATATGACGCGACGACTCGACGGTAAG-3'(配列番号79)
5'-CCGCTCGAGTTACGGGGTGGTGAAGCCGGCGTC-3' (配列番号80)
得られた822bpのrevG遺伝子断片を制限酵素(NdeI 及び XhoI)で切断後、大腸菌異種発現に用いるpET28b(+)b(ポリヒスチジン融合タンパク質発現ベクター:Novagen)に導入しpET28b(+)-revGを作製した。
E. coli BL21 Star(商標) (DE3)にpET28b(+)-revGを導入した。カナマイシン(50 μgml-1)を含むTB培地中で、OD600が0.5になるまで28℃培養(200 ml)し、0.5 mM IPTGを添加して遺伝子発現を誘導した。7時間 28℃で培養後、大腸菌を遠心分離により回収した。その後、0.5mg リゾチームml-1及び125 U ベンゾナーゼを含む20ml 緩衝液A (100mM NaH2PO4 (pH 7.8), 500mM NaCl, 5 mM イミダゾール, 10% グリセロール)に懸濁し超音波破砕を行った。遠心分離後、上清をNi-NTA (2 × 2 cm) (Qiagen)カラムに添着した。その後、0.2% Tween 20を含む緩衝液A (50 ml)、40 mM イミダゾールを含む緩衝液A (50 ml)で洗浄後、250 mM イミダゾールを含む緩衝液A (25 ml)でRevG (12 mg) を溶出した。その後、緩衝液B (50 mM NaH2PO4(pH 7.5), 100mM NaCl, 1 mM DTT, 10% 緩衝液) で透析を行い、アミコンウルトラセル30Kで濃縮し精製酵素RevG(7 mg ml-1)を調製した。
以下の最適化条件でスピロケタール環化酵素(RevG)反応を行った。
50mM glycine-NaOH (pH10), 1 mM DTT, 1 mM NAD+, 10% glycerol, 0.05 mM RM-A1aを含む溶液(100μl)を30℃ で5 分保温したのちに、2.8 pmol 精製酵素( RevG)を添加し反応を開始した。インキュベーション後43μl アセトニトリルを添加して反応を停止した。20,000 × g 遠心分離後に上清を回収し反応産物(20 μl)をLC/ESI-MSにて解析した。
反応産物をSep-Pak PLUS C18 columnにかけ、30%アセトニトリルで洗浄後、100%アセトニトリルで溶出して反応産物を得た。その結果、図6(a)、(c)、(d)に示すように、反応1分ではRM-A2aが生成し、反応10分ではRM-A3a及びRM-A3bが生成した。このことから、非環化状態のRM-A1aから、反応中間体RM-A2a、さらに不安定中間体 C15-dehydro-RM-A2aを経由して酸性HPLC条件下、スピロケタール環を有する RM-A3a及びRM-A3bに変換されることが判明した。
(1)revJ遺伝子の大腸菌異種発現ベクター作製
revJ遺伝子を含む鋳型フォスミド11A02、下記のプライマー、およびPrimeSTAR HS DNA polymerase (TaKaRa)を用い、98℃ 10秒後、98℃ 10 秒, 62℃ 5秒, 68℃ 1.5分の25サイクルPCR反応を行った。
5'-GGAATTCCATATGGTGACCGAGACCGAACAGCTC-3'(配列番号81)
5'-CCGCTCGAGTCAGACCCGGGTGAGGTCGAC-3'(配列番号82)
得られたrevJ遺伝子断片を制限酵素(NdeI 及び XhoI)で切断後、大腸菌異種発現に用いるpET28b(+)b(ポリヒスチジン融合タンパク質発現ベクター:Novagen)に導入しpET28b(+)-revJを作製した。
E. coli BL21 Star(商標) (DE3)にpET28b(+)-revJを導入した。カナマイシン(50 μgml-1)を含むTB培地中で、OD600が0.5になるまで28℃培養(200 ml)し、0.5 mM IPTGを添加して遺伝子発現を誘導した。7時間 28℃で培養後、大腸菌を遠心分離により回収した。その後、0.5mg リゾチームml-1及び125 U ベンゾナーゼを含む20ml 緩衝液A (100mM NaH2PO4 (pH 7.8), 500mM NaCl, 5 mM イミダゾール, 10% グリセロール)に懸濁し超音波破砕を行った。遠心分離後、上清をNi-NTA (2 × 2 cm) (Qiagen)カラムに添着した。その後、0.2% Tween 20を含む緩衝液A (50 ml)、40 mM イミダゾールを含む緩衝液A (50 ml)で洗浄後、250 mM イミダゾールを含む緩衝液A (25 ml)でRevJ (12 mg) を溶出した。その後、緩衝液B (50 mM NaH2PO4(pH 7.5), 100mM NaCl, 1 mM DTT, 10% 緩衝液) で透析を行い、アミコンウルトラセル30Kで濃縮し精製酵素RevJ(7 mg ml-1)を調製した。
RevG と RevJカップリング反応を以下の組成で行った。50 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 mM NAD+, 1 mM NADPH, 50 μM FAD, 0.05 mM RM-A1a, 2.96 nmol 精製RevG及び 2.93 nmol 精製RevJを含む溶液(100μl)を30℃ で20分反応した。20,000 × g 遠心分離後に上清を回収し反応産物(10 μl)をLCESI-MSにて0.05%ギ酸存在下(図6f)と非存在下(図6e)で解析した。
その結果、RevJが存在する場合のみ、最終生合成産物(リベロマイシンA)のスピロアセタール構造と一致する15S体のみが生成することから、RevJは立体制御因子であることが判明した。
HL-60 細胞(ヒト急性前骨髄性白血病細胞株)、K562 細胞(ヒト慢性骨髄性白血病細胞株)は,RPMI1640 medium(Invitrogen製)に10% Fetal Bovine Serum(Nichirei製)、0.5% penicilin/streptomycin溶液 (Invitrogen製)を添加した培地中、37℃、5% CO2の湿気中培養下で維持した。tsFT210 細胞(マウス乳がん細胞CDC2温度感受性株)は,RPMI1640 medium(Invitrogen製)に5% calf serum(Hyclone製)、0.5% penicilin/streptomycin溶液を添加した培地中、32℃、5% CO2の湿気中培養下で維持した。
HL-60細胞、K562細胞は96穴プレート(IWAKI製)に1.5×104cells/well/100 μlとなるように播種した。tsFT210細胞は96穴プレートに1.6×104cell/well/100μlとなるように播種した。表5の各薬剤を0.5%(v/v)で添加し、HL-60細胞、K562細胞を37℃、5% CO2の湿気中培養下、tsFT210細胞を32℃、5% CO2の湿気中培養下で維持した。薬剤添加から48時間後に、生細胞数測定試薬SF溶液(WST-8試薬、Nakarai tesque製)を各ウェルに10 μl添加し、HL-60細胞、K562細胞を37℃、5% CO2の湿気中培養下で30分、tsFT210細胞を32℃、5% CO2の湿気中培養下で1時間維持した。反応後、マイクロプレートリーダー (PerkinElmer製)を用いて、450 nmの吸光度を測定し、測定値から細胞増殖率を求めた。
大腸菌(HO141株)は、0.5% Polypeptone、0.5% Meat Extract、0.3% NaCl、0.001% Sodium Dodecyl Sulfate(SDS)の培地中、37℃で前培養した。600 nmの吸光度を測定し、吸光度が0.005となるように調製した大腸菌溶液を、96穴プレートに100 μlずつ播種した。表5の各薬剤を0.5%(v/v)で添加し、37℃で維持した。薬剤添加から6時間後に、マイクロプレートリーダー(PerkinElmer製)を用いて、600 nmの吸光度を測定し、測定値から増殖率を求めた。
出芽酵母(MLC30M株)は、2% Polypeptone、1% Yeast Extract、2% Glucose、0.02% Adenine、0.001% Sodium Dodecyl Sulfate(SDS)の培地中、30℃で前培養した。吸光度を測定し、吸光度が0.05となるように調製した酵母溶液を、96穴プレートに100 μlずつ播種した。表5の各薬剤を0.5%(v/v)で添加し、30℃で維持した。薬剤添加から18時間後に、マイクロプレートリーダー(PerkinElmer製)を用いて、600 nmの吸光度を測定し、測定値から増殖率を求めた。
5週齢雄ddYマウス(日本SLC製)の大腿骨および脛骨から骨髄細胞を採取し、α-MEM medium(Sigma-Aldrich製)に10% fetal bovine serum、0.5% penicilin/streptomycin溶液、50 ng/ml human M-CSF(ロイコプロール、協和発酵製)、1 ng/ml human TGF-β1(R&D Systems製)を添加した培地中、タイプIコラーゲンコートプレート(IWAKI製)に播種し、37℃、5% CO2の湿気中培養下で3日間維持した。その後、細胞をPBSで2回洗浄後、プレートに接着している細胞を骨髄マクロファージ細胞として使用した。骨髄マクロファージ細胞を、さらにα-MEM mediumに10% fetal-bovine serum、0.5% penicilin/streptomycin溶液、50 ng/ml human M-CSF、50 ng/ml human soluble RANKL(Peprotech製)を添加した培地中、37℃、5% CO2の湿気中培養下で3日間維持し、破骨細胞へ分化させた。
破骨細胞に表5の各薬剤を0.5%(v/v)で添加し、37℃、5% CO2の湿気中培養下で24時間維持した。その後、細胞を3.7% formalinを含むPBS溶液で室温下30分反応させ、溶液を除去後、さらにacetone/ethanol溶液(1:1 vol/vol)で室温下1分反応させ、溶液を除去して乾燥させた。固定化細胞にTRAP溶液 [50 mM sodium tartrate、90 mM sodium acetate、0.01% naphthol AS-MX phosphate(Sigma製)、0.05% fast red violet LB salt(Sigma製)、pH 5.0] で室温下30分反応させ、その後蒸留水で洗浄した。TRAP陽性多核破骨細胞数をカウントし、生存率を求めた。
酵素反応溶液 [20 mM imidazole, pH 7.5、75 mM MgCl2、0.5 mM DTT、1 U/ml tRNA(E. coli由来、Sigma製)、3 mM ATP、1μM isoleucine、10μCi/ml [3H]isoleucine(GE Healthcare製)、10μg protein(HT1080 cell lysate)]に、表5の各薬剤を1%(v/v)で添加し、合計量100μlで25℃、20分間反応させた。その後、1 mg/ml BSA溶液(400μl)および10% TCA溶液(500μl)を添加して反応を止め、4℃で一晩静置させた。遠心操作で得た沈殿物をGF-Cフィルター(Whatman製)上に移し、5% TCA溶液で3回洗浄後、フィルターを乾燥させた。バイアルに2 ml aquasol-2(PerkinElmer製)とフィルターを入れ、激しく攪拌後、[3H]isoleucine量を液体シンチレーションカウンター(Beckman製)で測定し、酵素活性率を求めた。
上記5~9の結果を表5にまとめた。その結果、RM-TはRM-Aに比べより高い癌細胞増殖阻害活性と標的分子IRSに対する酵素阻害活性を示した。このことから、RM-TはRM-Aよりも強い抗癌作用を発揮することが期待される。また、RM-Tメチルエステル、RM-Tエチルエステル、およびRM-Eも高い癌細胞増殖阻害活性を示した。さらに、RM-Tは酵母に対する増殖阻害作用を示し、抗真菌剤として使用できることが分かった。さらに、RM-Eは破骨細胞に対する生存阻害作用を示し、骨疾患治療剤として使用できることが分かった。
RM-Eの調製
野生株(Streptomyces sp. SN-593)をSY培地で2日間培養後、前培養液1 mlをRM-PM (70 ml)培地に植菌し、更に5日間培養を行った。その後、計3 Lの培養液に対して等量のアセトンを加え撹拌後、アセトンを除去し、酢酸でpHを4に調整し、等量の酢酸エチルを添加して抽出を2回行った。
その後、シリカゲルカラムクロマトグラフィーにより、クロロホルム/メタノール(10:1)画分を回収し、C18-HPLC(アセトニトリル:0.05%ギ酸=47:53)により8.49 mgを精製した。
野生株(Streptomyces sp. SN-593)をSY培地で2日間培養後、前培養液200 mlをRM-PM (14 L)培地に植菌しジャーファーメンターで4日間培養を行った。その後、等量のアセトンを加え撹拌後、アセトンを除去し、酢酸でpHを4に調整し、等量の酢酸エチルを添加して抽出を2回行った。C18-MPLCクロマトグラフィー後、Pegasil ODSを用いて0.05%ギ酸-アセトニトリル55-100%グラジェント溶出を行い、0.33 mgを精製した。
Org. Lett. 2005, 7(25):5573-5576. 清水らにより合成された。
野生株(Streptomyces sp. SN-593)をSY培地で2日間培養を行った。その後、前培養液1 ml をRM-PM (70 ml)培地に植菌し培養を行った。3日後、終濃度で1%になるようにエタノールを加え、さらに2日間培養を行った。その後、合計1.4 Lの培養液に等量のアセトンを加え撹拌後、アセトンを除去し、酢酸でpHを4に調整し、等量の酢酸エチルを添加して抽出を2回行った後、酢酸エチルを除去し、1.2gの粗画分を得た。ヘキサン/酢酸エチル/酢酸(100:100:1)溶媒を用いてシリカゲルカラムクロマト後にC18-HPLC精製を行いRM-T ethylesterを得た。RM-T methylesterの調製は、上記と同様であるが、エタノールの代わりにメタノールを添加して培養した。
RevHの大量発現と機能解析
(1) revH遺伝子の大腸菌異種発現ベクター作製
大腸菌による異種発現を容易にするために、revH遺伝子はオペロンバイオテクノロジー株式会社の人工遺伝子合成サービスにより配列を最適化して合成した。合成した配列(制限酵素配列を含む)を配列番号99に示す。
合成されたrevH遺伝子断片を制限酵素(NdeI及びXhoI)で切断後、大腸菌異種発現に用いるpET28b(+)(ポリヒスチジン融合タンパク質発現ベクター:Novagen)に導入しpET28b(+)-revHを作製した。
E. coli BL21 Star (商標) (DE3) にpET28b(+)-revHを導入した。カナマイシン(50 μg ml-1)を含むTB培地中でOD600が1.5になるまで37℃で培養(1l)し、0.1 mM IPTGを添加して遺伝子発現を誘導した。16時間18℃で培養後、大腸菌を遠心分離により回収した。その後Lysis Buffer (Wash Buffer + 1% Tween 20)に懸濁し、超音波破砕を行った。遠心分離後、上清をNi-NTA(2×2 cm)(Qiagen) カラムに吸着した。その後、50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 20 mM イミダゾール, 20% グリセロールで洗浄後、50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 250 mM イミダゾール, 20% グリセロールで溶出した。その後アミコンウルトラセル30Kを用い、50 mM Tris-HCl (pH 8.0), 0.1 M NaClでバッファー交換後、濃縮しRevHの精製酵素 (5 mg ml-1, 2 ml)を調製した。
以下の反応溶液組成でRM-A8aを調製した。
50 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 1 mM NAD+, 0.7 mM NADPH, 0.1 mM FAD, 0.04 mM RM-A1a, 100 nmol 精製RevG及び31 nmol 精製RevHを含む溶液(30 ml)を30℃で150分間反応した。次に20 nmolの精製RevJを添加しさらに30℃で120分間反応を行った。反応終了後反応溶液を等量の酢酸エチルで2回抽出し、硫酸ナトリウムで脱水した後、酢酸エチルをエバポレーターで溜去した。残渣をメタノールに溶解し、HPLC (カラム:PEGASIL ODS (20 mm× 250 mm, 株式会社センシュー化学)に供し、分取した。溶出は85%アセトニトリル、流速8 ml min-1で行い、溶出時間は25分であった。さらにアセトニトリル/水をエバポレーターで溜去し、約0.3 mgのRM-A8aを得た。
RM-A6a とRM-A9aはrevE破壊株により生産される(図14)。
revE遺伝子破壊株をSY培地で2日間培養を行った後、この前培養液1 mlをRM-PM (70 ml)培地に植菌し本培養を5日間行った。合計5 Lの培養液に等量のアセトンを加え代謝産物の抽出、アセトン除去、酢酸でpHを4に調整を行った。次に、等量の酢酸エチルを添加して3回抽出を行い、酢酸エチルを除去し3 gの粗画分を得た。その後、シリカゲルカラムクロマトグラフィーにより、クロロホルム/メタノール (10: 5)画分を回収し、C18-HPLC (アセトニトリル: 0.05%ギ酸=60: 40)精製を行った。更にC18-HPLC (アセトニトリル: 0.05%ギ酸=75: 25)により6.9 mgのRM-A6aを精製した。RM-A6aと同一の粗画分から、シリカゲルカラムクロマトグラフィーにより、クロロホルム/メタノール (10: 5)画分を回収し、C18-HPLC (アセトニトリル: 0.05%ギ酸=60: 40)精製によりRM-A9aを含む画分130 mgを得た。更にこれをC18-HPLC (0.05%ギ酸-アセトニトリル 60-100%グラジェント溶出、C18-HPLC (アセトニトリル: 0.05%ギ酸=52: 48)精製により18 mgのRM-A9aを精製した。
Claims (16)
- リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌であって、配列番号36のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードするrevQ遺伝子の発現が親株と比較して増大するように改変され、それにより前記生産能が該親株よりも向上したストレプトマイセス属細菌。
- revQ遺伝子のコピー数を高めること、またはrevQ遺伝子のプロモーターの改変によってrevQ遺伝子の発現が増大した、請求項1に記載の細菌。
- ストレプトマイセス sp. SN-593株をrevQ遺伝子の発現が増大するように改変することによって得られる、請求項1または2に記載の細菌。
- 請求項1~3のいずれか一項に記載のストレプトマイセス属細菌を培地で培養し、培地中にリベロマイシンAまたはその合成中間体を蓄積させ、その培養物からリベロマイシンAまたはその合成中間体を回収することを含む、リベロマイシンAまたはその合成中間体の製造方法。
- 配列番号36のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードし、リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌に導入したときに、該生産能を向上させるポリヌクレオチド。
- 配列番号35の121-951の塩基配列とストリンジェントな条件でハイブリダイズし、リベロマイシンAまたはその合成中間体の生産能を有するストレプトマイセス属細菌に導入したときに、該生産能を向上させるポリヌクレオチド。
- 前記RevGタンパク質とともに配列番号20のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列を有するRevJタンパク質を化合物(I)に作用させる、請求項7に記載の化合物(II)の製造方法。
- 配列番号14のアミノ酸配列または該アミノ酸配列と80%以上の同一性を有するアミノ酸配列を有し、化合物(I)を化合物(II)に変換する反応を触媒する活性を有するタンパク質。
- 請求項10に記載のタンパク質をコードするポリヌクレオチド。
- 配列番号13の121-939の塩基配列の相補鎖とストリンジェントな条件でハイブリダイズする、請求項11に記載のポリヌクレオチド。
- 配列番号2、4、6、8、10、12、16、18、20、22、24、26、28、30、34、38、40、42または44のアミノ酸配列と80%以上の同一性を有するアミノ酸配列をコードし、リベロマイシン生合成関連タンパク質をコードするポリヌクレオチド。
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