WO1997029205A1 - Effective production method of be-13793c - Google Patents

Effective production method of be-13793c Download PDF

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Publication number
WO1997029205A1
WO1997029205A1 PCT/US1997/001896 US9701896W WO9729205A1 WO 1997029205 A1 WO1997029205 A1 WO 1997029205A1 US 9701896 W US9701896 W US 9701896W WO 9729205 A1 WO9729205 A1 WO 9729205A1
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Prior art keywords
medium
strain
agar
culture
streptomyces
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PCT/US1997/001896
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French (fr)
Inventor
Bruce W. Burgess
Randolph L. Greasham
Chanyong Lee
Wen-Jun Sun
Hiroyuki Suda
Kenji Kawamura
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Banyu Pharmaceutical Co., Ltd.
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Priority to AU22608/97A priority Critical patent/AU2260897A/en
Publication of WO1997029205A1 publication Critical patent/WO1997029205A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to an effective production method of BE-13793C, 12,13-dihydro-l,ll-dihydroxy-5H-indolo [2 ,3- a]pyrrolo[3, -c]carbazole-5,7 (6H)-dione, which is an important compound useful in the medical fields, more particularly for a starting material for preparing indolopyrrolocarbazole antitumor substances.
  • the present inventors had done the screening of antitumor substances among microbial products and found a new antitumor agent, BE-13793C (12, 13-dihydro-l, ll-dihydroxy-5H-indolo [2, 3- a]pyrrolo[3, 4-c]carbazole-5, 7 (6H) -dione) , and disclosed in Japanese Patent Application Laying Open No. 3-20277 (1991) (refer to J. Antibiotics 44, 723-728 (1991)) .
  • indolopyrrolocarbazole derivatives in which a monosaccharide residue was introduced to the position 13 of BE-13793C showed better antitumor activities than the parent compound (refer to WO91/18003) .
  • indolopyrrolocarbazole compounds which had a monosaccharide residue at position 13 and one of various substituents at position 6 of BE-13793C were found to show more excellent antitumor activities as disclosed in European Patent Application No. EP-A-545195.
  • BE-13793C has been verified as a very important intermediate for the synthesis of antitumor indolopyrrolocarbazole compounds. Therefore, an effective production method of BE-13793C is strongly desired in the field of production of antitumor medicaments.
  • BE-13793C is produced by a strain of the genus S rept-nmynpg as disclosed in U.S. Patent No. 5,290,698.
  • the production output of BE-13793C by the strain was relatively low; for example, the strain BA-13793 produced about 40 ⁇ g/ml of BE-13793C.
  • the strain BA-13793 produced about 40 ⁇ g/ml of BE-13793C.
  • the present inventors have investigated mutants of the strain and found that a mutant strain BA-13793-725M or BA-13793- 651N may produce BE-13793C in an amount significantly higher than the productivity of the parent strain. Further, it has been found that the mutant BA-13793-725M or BA-13793-651N may produce BE- 13793C in still higher amounts when cultured in an improved fermentation medium of the instant invention.
  • the present invention provides S reptomyces sp. BA-13793-725M or BA-13793-651N which can .produce BE-13793C in a higher amount than St-reptonv, .:es sp. BA-13793.
  • the present invention also provides a method for effectively producing BE-13793C by culturing the strain S reptomyces sp. BA- 13793-725M or BA-13793-651N. Further, an improved culture medium is provided which is suitable for the fermentation of the strains BA-13793-725M and BA- 13793-651N to effectively produce BE-13793C.
  • the mutant microoganism strains of the present invention may be derived from the strain Streptomyces sp. BA-13793 in a conventional manner such as screening of higher BE-13793C-producing strains, treatment with a chemical mutagen (e.g., N-methyl-N'- nitro-N-nitrosoguanidine or NTG) , irradiation with ultraviolet light, or any combination thereof.
  • a chemical mutagen e.g., N-methyl-N'- nitro-N-nitrosoguanidine or NTG
  • irradiation with ultraviolet light or any combination thereof.
  • One of the mutants derived from Streptomyces sp. BA-13793 has the following bacteriological characteristics.
  • the strain was cultured in a Pridham-Gottling agar base medium to which the following sugars were added at 28°C for 14 days.
  • D-glucose + raffinose: D-xylose: - D-mannitol L-arabinose : inositol: L-rhamnose : salicin: D-fructose- + sucrose: D-galactose: +
  • strain Streptomyces sp. BA-13793-725M has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5237 on September 21, 1995.
  • Another mutant derived from Streptomyces sp. BA-13793 has the following bacteriological characteristics.
  • the strain was cultured at 28°C for 14 days in a Pridham- Gottling agar base medium to which the following sugars were added.
  • strain Streptomyces sp. BA-13793-651N has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5326 on December 5, 1995.
  • the improved strain of S reptomyces sp is cultured in a nutrient medium under aerobic conditions.
  • Various known carbon and nitrogen sources which are commonly used in the culture of Ac inomycetes may be used as the nutrients.
  • carbon sources commercially available glucose, maltose, starch, saccharose, molasses, dextrin or any combination thereof can be used, but glucose and dextrin are preferred.
  • nitrogen sources commercially available soy bean meal, corn gluten meal, corn steep liquor, meat extract, yeast extract, dried yeast, cotton seed powder, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat bone powder, inorganic ammonium salts, sodium nitrate or any mixture thereof may be used for the culture of the producing strain.
  • corn gluten meal, yeast extract and fish meal can be used particularly effectively.
  • inorganic salts commercially available calcium carbonate, calcium chloride, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate and/or various phosphate salts can be used.
  • heavy metal ions such as iron, copper, manganese, zinc, cobalt and/or molybdate can optionally be added to the culture medium in a small amount.
  • Sodium chloride, magnesium sulfate, calcium chloride, ferrous sulfate, cupric chloride, manganese chloride and ammonium molybdate are preferred.
  • plant oils such as soy bean oil and linseed oil, higher alcohols such as octadecanol, or various silicone compounds can optionally be used.
  • the strain may be cultured in the same manner as described in U.S. Patent No. 5,290,698.
  • the culture methods of the producing strain may be either solid or liquid culture.
  • any of standing culture, stirring culture, shaking culture and aeration culture may be used.
  • shaking culture or aeration culture is preferably used.
  • the temperature for culture may conveniently be 15 to 32°C, but 20 to 30°C is preferable.
  • the optimum pH for the culture is in the range of 4 to 8 and the culture time required is between 2 days and 21 days, but a period of 5 days to 15 days is preferable.
  • the present invention further relates to.improved culture media for use in the culture of the improved strain to produce BE- 13793C in still higher amounts.
  • Examples of the improved culture media are illustrated in Examples 2 (5) and 3 which follow. As seen from the Examples, the productivity attained was several times higher than that obtained from the culture in conventional media.
  • Streptomyces sp. BA-13793 grown on an agar slant was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium consisting of glucose 0.1%, dextrin 2.0%, corn gluten meal 1.0%, fish meal 0.5%, yeast extract 0.1%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008%, ammonium molybdate 0.00024% and 3- (N-morpholino)propane- sulfonic acid 0.5% (pH 6.7) and the flask was cultured on a rotary shaker (180 rpm) at 28°C for 72 hours.
  • BE-7 medium consisting of glucose 0.1%, dextrin 2.0%, corn gluten meal 1.
  • One ml of the culture broth was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of the BE-7 medium and the flask was cultured on a rotary shaker (180 rpm) at 28°C for 120 hours.
  • Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction.
  • the concentration of BE-13793C in the extract was measured by HPLC to be 40 ⁇ g/ml of the culture broth.
  • the spores of Streptomyces sp. BA-13793 cultured on an ISP-4 agar slant medium consisting of soluble starch 1.0%, ammonium sulfate 0.2%, monopotassium phosphate 0.1%, sodium chloride 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.2% and agar 2.0% were suspended in 10 ml of sterilized water and 100, 1,000, 10,000 and 100,000 times diluted solutions were made with sterilized water. Each diluted solution (0.2 ml) was spread on an ISP-4 agar plate and the plate was incubated at 28°C for 6 days. Colonies that appeared on the agar plates were transferred to ISP-4 agar slant medium and cultured at 28°C for 14 days. Each colony was cultured by the method described in Example 1 (2) and high BE- 13793C producing strains were selected.
  • Example 2 (1) , (2) and (3) were repeated in the following order M ⁇ M ⁇ N ⁇ N ⁇ M ⁇ N ⁇ U ⁇ N- ⁇ M ⁇ N ⁇ N ⁇ M and high BE-13793C producing strains were obtained. Two of these mutant strains were designated as BA-13793-651N and BA-13793-725M.
  • the BE-13793C productivity of Streptomyces sp. BA-13793-725M by the method described in Example 1 (2) was 1,420 ⁇ g/ml of culture broth. This value is 17.8 times that obtained in Example 1 (2) for the parent BA-13793 strain.
  • the mutant strains Streptomyces sp. BA-13793-725M and BA- 13793-651N had the bacteriological characteristics as hereinbefore described. These strains Streptomyces sp. BA-13793-725M and BA- 13793-651N were deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5237 on September 21, 1995 and BP-5326 on December 5, 1995, respectively.
  • One ml of the culture broth was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008%, ammonium molybdate 0.00024% and 3- (N- morpholino)propanesulfonic acid 0.5% (pH 6.7) and the flask was cultured on a rotary shaker (180 rpm) at 25°C for 14 days.
  • a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish
  • Example 1 Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction.
  • the concentration of BE-13793C in the extract was measured by HPLC to be 5,400 ⁇ g/ml of the culture broth. This value is 135 times that obtained in Example 1 (1) for the parent BA-13793 and 3.8 times that obtained in Example 2 (4) .
  • the mutant strain Streptomyces sp. BA-13793-725M grown on an agar slant medium was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium (pH 6.7) described in Example 1 (1) and cultured at 28°C for 72 hours on a rotary shaker (180 rpm) .
  • One hundred ml of the culture broth was inoculated into a 20-L fermentor containing 10 L of BE-7 medium (pH 6.7) and cultured at 28°C for 48 hours with aeration of 10 L/min and 200 rpm agitation.
  • Two L of the culture broth were inoculated into a 200-L fermentor containing 100 L of a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008% and ammonium molybdate 0.00024% (pH 6.7) and fermentation was performed at 25°C for 14 days with aeration of 100 L/min and 180 rpm agitation.
  • a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride
  • One hundred twenty ml of the culture broth was inoculated into a 23-L fermentor containing 12 L of a medium consisting of glucose 0.2%, maltodextrin 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15% and antifoaming agent P-2000 0.4% (pH 7.0) and cultured at 25°C for 15 days with aeration of 12 L/min and 600 rpm agitation. During this fermentation, the pH of the culture medium was maintained below 7.0. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 6, 600 ⁇ g/ml of the culture broth.
  • THF tetrahydrofuran

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Abstract

Disclosed herein is an improved method for effectively producing BE-13793C, 12,13-dihydro-1,11-dihydroxy-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, by a fermentation process using a microorganism. The compound BE-13793C has itself an antitumor activity and may be used as a starting material for preparing more excellent antitumor substances. The effective production method is expected to be applicable in the field of production of medical drugs.

Description

FFFFΓTTVF PROΠΠΓTTON METHOD OF BE-1 7930
FTF.T.D OF THF. INVFNTTON
The present invention relates to an effective production method of BE-13793C, 12,13-dihydro-l,ll-dihydroxy-5H-indolo [2 ,3- a]pyrrolo[3, -c]carbazole-5,7 (6H)-dione, which is an important compound useful in the medical fields, more particularly for a starting material for preparing indolopyrrolocarbazole antitumor substances.
ΑC πROrT D OF THF. TNVFNTTON
The present inventors had done the screening of antitumor substances among microbial products and found a new antitumor agent, BE-13793C (12, 13-dihydro-l, ll-dihydroxy-5H-indolo [2, 3- a]pyrrolo[3, 4-c]carbazole-5, 7 (6H) -dione) , and disclosed in Japanese Patent Application Laying Open No. 3-20277 (1991) (refer to J. Antibiotics 44, 723-728 (1991)) . Thereafter, it was found that indolopyrrolocarbazole derivatives in which a monosaccharide residue was introduced to the position 13 of BE-13793C showed better antitumor activities than the parent compound (refer to WO91/18003) . Moreover, indolopyrrolocarbazole compounds which had a monosaccharide residue at position 13 and one of various substituents at position 6 of BE-13793C were found to show more excellent antitumor activities as disclosed in European Patent Application No. EP-A-545195. Thus, BE-13793C has been verified as a very important intermediate for the synthesis of antitumor indolopyrrolocarbazole compounds. Therefore, an effective production method of BE-13793C is strongly desired in the field of production of antitumor medicaments.
BE-13793C is produced by a strain of the genus S rept-nmynpg as disclosed in U.S. Patent No. 5,290,698. However, the production output of BE-13793C by the strain was relatively low; for example, the strain BA-13793 produced about 40 μg/ml of BE-13793C. Thus, there is a need for improved mutant or variant microorganisms and more effective method for producing BE-13793C.
SUMMARY OF THE INVENTION
To improve the productivity of the BE-13793C-producing strain BA-13793, the present inventors have investigated mutants of the strain and found that a mutant strain BA-13793-725M or BA-13793- 651N may produce BE-13793C in an amount significantly higher than the productivity of the parent strain. Further, it has been found that the mutant BA-13793-725M or BA-13793-651N may produce BE- 13793C in still higher amounts when cultured in an improved fermentation medium of the instant invention.
Accordingly, the present invention provides S reptomyces sp. BA-13793-725M or BA-13793-651N which can .produce BE-13793C in a higher amount than St-reptonv, .:es sp. BA-13793.
The present invention also provides a method for effectively producing BE-13793C by culturing the strain S reptomyces sp. BA- 13793-725M or BA-13793-651N. Further, an improved culture medium is provided which is suitable for the fermentation of the strains BA-13793-725M and BA- 13793-651N to effectively produce BE-13793C.
DESCRIPTION OF THE INVENTION
The mutant microoganism strains of the present invention may be derived from the strain Streptomyces sp. BA-13793 in a conventional manner such as screening of higher BE-13793C-producing strains, treatment with a chemical mutagen (e.g., N-methyl-N'- nitro-N-nitrosoguanidine or NTG) , irradiation with ultraviolet light, or any combination thereof.
One of the mutants derived from Streptomyces sp. BA-13793 has the following bacteriological characteristics.
1. Morphology:
As compared with the parent strain, the formation of aerial hyphae is poor. Whirls are formed like the parent strain, but the number thereof is small and few spores are observed. Further, neither any special organ, such as sporangia, flagellated spores or sclerotia, nor fragmentation of hyphae is observed, like the parent strain.
2. Cultural characteristics:
The cultural characteristics on various agar plate media at 28°C for 14 days are shown below. Color of Soluble
Medium Growth Aerial Hypha Basal Hypha Pigment
Yeast-malt- good trace deep brown none agar (ISP-2)
Oat eal-agar good trace deep brown none (ISP-3)
Starch-in¬ moderate poor deep orange none organic salt- grayish agar (ISP-4) white
Glycerol- moderate none deep orange none asparagine (ISP-5)
Tyrosine- moderate poor brownish none agar (ISP-7) grayish orange white
Nutrient good none deep brown none agar
Sucrose- moderate trace light none nitrate- orange agar
Glucose- poor none light none asparagine- orange agar
3. Growth temperature (starch-inorganic salt-agar, 14 clays) : 11°C: No growth;
16°C Moderate growth and no formation of aerial hypha; 19°C Good growth and no formation of aerial hypha; 23°C Good growth and poor formation of aerial hyphae; 28°C Good growth and poor formation of aerial hyphae; 31°C Good growth and poor formation of aerial hyphae; 35°C Moderate growth and no formation of aerial hypha; 40°C No growth . 4. Physiological characteristics:
(1) Liquefaction of gelatin: positive (glucose-peptone-gelatin medium)
(2) Hydrolysis of starch: positive
(starch-inorganic salt-agar medium)
(3) Coagulation of skim milk: negative (skim milk medium)
(4) Peptonization of skim milk: positive
(skim milk medium)
(5) Production of melanoid pigments: negative (ISP-1, ISP-6, ISP-7)
(6) Resistance to common salt: growing at a common salt content below 2%
(starch-inorganic salt-agar medium)
5. Utilization of carbon sources:
The strain was cultured in a Pridham-Gottlieb agar base medium to which the following sugars were added at 28°C for 14 days.
D-glucose: + raffinose: D-xylose: - D-mannitol L-arabinose : inositol: L-rhamnose : salicin: D-fructose- + sucrose: D-galactose: +
6. Composition of cell wall:
LL-diaminopimelic acid and glycine were detected. Thus, this strain is poor in adhesion of aerial hyphae as compared with the parent strain Streptomyces sp. BA-13793 and typical whirls are not well observed. As a result, this strain is designated as Streptomyces sp. BA-13793-725M.
The strain Streptomyces sp. BA-13793-725M has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5237 on September 21, 1995.
Another mutant derived from Streptomyces sp. BA-13793 has the following bacteriological characteristics.
1. Morphology:
As compared with the parent strain, the formation of aerial hyphae is poor. Whirls are formed like the parent strain, but the number thereof is small and few spores are observed. Further, neither any special organ, such as sporangia, flagellated spores or sclerotia, nor fragmentation of hyphae is observed, like the parent strain.
2. Cultural characteristics:
The cultural characteristics on various agar plate media at 28°C for 14 days are shown below. Color of Soluble
Medium Growth Aerial Hypha Basal Hypha Piσment
Yeast-malt- good none deep brown none agar (ISP-2)
Oatmeal-agar good trace deep brown none (ISP-3)
Starch-in¬ good moderate deep orange none organic salt- grayish agar (ISP-4) white
Glycerol- moderate poor brownish none asparagine grayish orange
(ISP-5) white
Tyrosine- moderate poor brownish none agar (ISP-7) grayish orange white
Nutrient moderate none deep brown none agar
Sucrose- poor trace light none nitrate- orange agar
Glucose- moderate trace light none asparagine- orange agar
3. Growth temperature (starch-inorganic salt-agar, 14 days) : 10°C: No growth;
15°C Moderate growth and no formation of aerial hypha; 18°C Moderate growth and poor formation of aerial hypha; 22°C Moderate growth and poor formation of aerial hypha; 26°C Good growth and moderate formation of aerial hypha; 29°C Good growth and moderate formation of aerial hypha; 32°C Moderate growth and moderate formation of aerial hypha; 37°C No growth . 4. Physiological characteristics:
(1) Liquefaction of gelatin: positive (glucose-peptone-gelatin medium)
(2) Hydrolysis of starch: positive (starch-inorganic salt-agar medium)
(3) Coagulation of skim milk: negative (skim milk medium)
(4) Peptonization of skim milk: positive
(skim milk medium)
(5) Production of melanoid pigments: negative (ISP-1, ISP-6, ISP-7)
(6) Resistance to common salt: growing at a common salt content below 2%
(starch-inorganic salt-agar medium)
5. Utilization of carbon sources:
The strain was cultured at 28°C for 14 days in a Pridham- Gottlieb agar base medium to which the following sugars were added. D-glucose: + raffinose :
D-xylose: - D-mannitol:
L-arabinose: - inositol: ±
L-rhamnose: ± salicin: ±
D-fructose: + sucrose:
D-galactose: +
6. Composition of cell wall:
LL-diaminopimelic acid and glycine were detected. Thus, this strain is designated as Streptomyces sp. BA-13793-
651N.
The strain Streptomyces sp. BA-13793-651N has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5326 on December 5, 1995.
In order to produce BE-13793C in high activity according to the present invention, the improved strain of S reptomyces sp . BA- 13793 is cultured in a nutrient medium under aerobic conditions. Various known carbon and nitrogen sources which are commonly used in the culture of Ac inomycetes may be used as the nutrients.
For example, as carbon sources, commercially available glucose, maltose, starch, saccharose, molasses, dextrin or any combination thereof can be used, but glucose and dextrin are preferred. As nitrogen sources, commercially available soy bean meal, corn gluten meal, corn steep liquor, meat extract, yeast extract, dried yeast, cotton seed powder, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat bone powder, inorganic ammonium salts, sodium nitrate or any mixture thereof may be used for the culture of the producing strain. Among these nitrogen sources, corn gluten meal, yeast extract and fish meal can be used particularly effectively.
As inorganic salts, commercially available calcium carbonate, calcium chloride, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate and/or various phosphate salts can be used. In addition, heavy metal ions such as iron, copper, manganese, zinc, cobalt and/or molybdate can optionally be added to the culture medium in a small amount. Sodium chloride, magnesium sulfate, calcium chloride, ferrous sulfate, cupric chloride, manganese chloride and ammonium molybdate are preferred. Further, as antifoaming agents, plant oils such as soy bean oil and linseed oil, higher alcohols such as octadecanol, or various silicone compounds can optionally be used. Other compounds which can be utilized by the improved strain to produce BE-13793C, such as 3- (N-morpholino)propanesulfonic acid and sodium borate, may be used for the culture .
The strain may be cultured in the same manner as described in U.S. Patent No. 5,290,698. The culture methods of the producing strain may be either solid or liquid culture. In case of liquid culture, any of standing culture, stirring culture, shaking culture and aeration culture may be used. Among them, shaking culture or aeration culture is preferably used. The temperature for culture may conveniently be 15 to 32°C, but 20 to 30°C is preferable. The optimum pH for the culture is in the range of 4 to 8 and the culture time required is between 2 days and 21 days, but a period of 5 days to 15 days is preferable.
The present invention further relates to.improved culture media for use in the culture of the improved strain to produce BE- 13793C in still higher amounts. Examples of the improved culture media are illustrated in Examples 2 (5) and 3 which follow. As seen from the Examples, the productivity attained was several times higher than that obtained from the culture in conventional media.
This invention will be explained in more detail by the following Examples; however, various modifications and/or changes may be made in the examples by those skilled in the art without departing from the spirit and scope of the invention as defined in the attached claims . Example 1
(1) Streptomyces sp. BA-13793 grown on an agar slant was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium consisting of glucose 0.1%, dextrin 2.0%, corn gluten meal 1.0%, fish meal 0.5%, yeast extract 0.1%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008%, ammonium molybdate 0.00024% and 3- (N-morpholino)propane- sulfonic acid 0.5% (pH 6.7) and the flask was cultured on a rotary shaker (180 rpm) at 28°C for 72 hours. One ml of the culture broth was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of the BE-7 medium and the flask was cultured on a rotary shaker (180 rpm) at 28°C for 120 hours. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 40 μg/ml of the culture broth.
(2) Streptomyces sp. BA-13793 grown on an agar slant was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium (pH 6.7) described in (1) and the flask was cultured on a rotary shaker (180 rpm) at 28°C for 72 hours. One ml of the culture broth was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium and the flask was cultured on a rotary shaker (180 rpm) at 25°C for 120 hours. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 80 μg/ml of the culture broth. This value is 2.0 times that obtained in (1) where the second culture was carried out at 28°C. Example 2
(1) Monospore culture of Streptomyces sp . BA-13793 (Method
M) .
The spores of Streptomyces sp. BA-13793 cultured on an ISP-4 agar slant medium (pH 7.0) consisting of soluble starch 1.0%, ammonium sulfate 0.2%, monopotassium phosphate 0.1%, sodium chloride 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.2% and agar 2.0% were suspended in 10 ml of sterilized water and 100, 1,000, 10,000 and 100,000 times diluted solutions were made with sterilized water. Each diluted solution (0.2 ml) was spread on an ISP-4 agar plate and the plate was incubated at 28°C for 6 days. Colonies that appeared on the agar plates were transferred to ISP-4 agar slant medium and cultured at 28°C for 14 days. Each colony was cultured by the method described in Example 1 (2) and high BE- 13793C producing strains were selected.
(2) Mutation of Streptomyces sp. BA-13793 by N-methyl-N* - nitro-N-nitrosoguanidine (NTG) (Method N) .
Spores of Streptomyces sp. BA-13793 grown on the ISP-4 agar slant medium (pH 7.0) described in Example 2 (1) were suspended in 10 ml of 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mg of NTG and the solution was shaken at 28°C for 30 minutes. The NTG treated solution was diluted 10, 100 and 1,000 times by sterilized water and colonies were obtained by the method described in Example 2 (1) . Each colony was cultured by the method described in Example 1 (2) and high BE-13793C producing strains were selected.
(3) Mutation of Streptomyces sp. BA-13793 by UV light irradiation (Method U) .
Spores of Streptomyces sp. BA-13793 grown on the ISP-4 agar slant medium (pH 7.0) described in Example 2 (1) were suspended in 10 ml of sterilized water and the suspension was transferred to a petri dish of 9 cm in diameter. The dish was irradiated by UV light for 30 seconds with stirring. The treated suspension was diluted 10, 100 and 1,000 times with sterilized water and colonies were obtained by the method described in Example 2 (1) . Each colony was cultured by the method described in Example 1 (2) and high BE-13793C producing strains were selected.
(4) Creation of mutants of Streptomyces sp. BA-13793.
The methods described in Example 2 (1) , (2) and (3) were repeated in the following order M→M→N→N→M→N→U→N-→M→N→N→M and high BE-13793C producing strains were obtained. Two of these mutant strains were designated as BA-13793-651N and BA-13793-725M. The BE-13793C productivity of Streptomyces sp. BA-13793-725M by the method described in Example 1 (2) was 1,420 μg/ml of culture broth. This value is 17.8 times that obtained in Example 1 (2) for the parent BA-13793 strain.
The mutant strains Streptomyces sp. BA-13793-725M and BA- 13793-651N had the bacteriological characteristics as hereinbefore described. These strains Streptomyces sp. BA-13793-725M and BA- 13793-651N were deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba, Japan under the Budapest Treaty with the accession number FERM BP-5237 on September 21, 1995 and BP-5326 on December 5, 1995, respectively.
(5) Improvement of production medium.
(5)-l The mutant strain Streptomyces sp. BA-13793-725M grown on an agar slant medium was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium (pH 6.7) described in Example 1 (1) and cultured at 28°C for 72 hours on a rotary shaker (180 rpm) . One ml of the culture broth was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008%, ammonium molybdate 0.00024% and 3- (N- morpholino)propanesulfonic acid 0.5% (pH 6.7) and the flask was cultured on a rotary shaker (180 rpm) at 25°C for 14 days. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 5,400 μg/ml of the culture broth. This value is 135 times that obtained in Example 1 (1) for the parent BA-13793 and 3.8 times that obtained in Example 2 (4) .
(5) -2 The mutant strain Streptomyces sp. BA-13793-651N grown on an agar slant medium was inoculated into a 250-ml Erlenmeyer flask containing 60 ml of BE-7 medium (pH 6.7) described in Example 1 (1) and cultured at 28°C for 72 hours on a rotary shaker (200 rpm) . One ml of the culture broth was inoculated into a 250-ml Erlenmeyer flask containing 60 ml of a medium consisting of glucose 0.2%, maltodextrin 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15% and antifoaming agent P-2000 0.4% (pH 7.0) and the flask was cultured on a rotary shaker (200 rpm) at 25°C for 12 days. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 5,400 μg/ml of the culture broth. Example 3
(1) The mutant strain Streptomyces sp. BA-13793-725M grown on an agar slant medium was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of BE-7 medium (pH 6.7) described in Example 1 (1) and cultured at 28°C for 72 hours on a rotary shaker (180 rpm) .
One hundred ml of the culture broth was inoculated into a 20-L fermentor containing 10 L of BE-7 medium (pH 6.7) and cultured at 28°C for 48 hours with aeration of 10 L/min and 200 rpm agitation. Two L of the culture broth were inoculated into a 200-L fermentor containing 100 L of a medium consisting of glucose 0.2%, corn dextrin (MS-3600) 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008% and ammonium molybdate 0.00024% (pH 6.7) and fermentation was performed at 25°C for 14 days with aeration of 100 L/min and 180 rpm agitation. During this fermentation, glucose solution was added to maintain the pH of the culture below 7.0. Total amount of glucose added was 3.9%. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes, followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 5,420 μg/ml of the culture broth.
(2) The mutant strain Streptomyces sp. BA-13793-651N grown on an agar slant medium was inoculated into two 250-ml Erlenmeyer flasks each containing 60 ml of BE-7 medium (pH 6.7) described in Example 1 (1) and cultured at 28°C for 72 hours on a rotary shaker (200 rpm) .
One hundred twenty ml of the culture broth was inoculated into a 23-L fermentor containing 12 L of a medium consisting of glucose 0.2%, maltodextrin 8.0%, corn gluten meal 1.5%, fish meal 6.0%, yeast extract 0.15% and antifoaming agent P-2000 0.4% (pH 7.0) and cultured at 25°C for 15 days with aeration of 12 L/min and 600 rpm agitation. During this fermentation, the pH of the culture medium was maintained below 7.0. Five ml of the culture broth was extracted with 50 ml of tetrahydrofuran (THF) and shaken for 30 minutes followed by extraction. The concentration of BE-13793C in the extract was measured by HPLC to be 6, 600 μg/ml of the culture broth.

Claims

WHAT TS CLAIMED TS:
1. A method of producing BE-13793C, 12, 13-dihydro-l, 11- dihydroxy-5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7 (6H) -dione, comprising culturing a strain Streptomyces sp. BA-13793-725M (FERM BP-5237) or BA-13793-651N (FERM BP-5326) .
2. A Strain S reptomγres sp. BA-13793-725M (FERM BP-5237) or BA-13793-651N (FERM BP-5326) .
3. The method according to Claim 1 wherein the cultuling is performed in a fermentation medium that is maintained at a temperature of about 25°C , but not more than 26°C.
4. The method according to Claim 3 wherein the culturing is performed in a fermentation medium that is maintained at a pH of less than about 7.0.
5. The method according to Claim 3 wherein the culturing is performed in a fermentation medium is that comprises the following ingredients :
8.0% dextrin source, and
6.0% fish meal; wherein the dextrin source is selected from corn dextrin (MS-3600) and maltodextrin. 6. The method according to Claim 5 wherein the culturing is performed in a fermentation medium that comprises the following ingredients:
0.2% glucose,
8.0% dextrin source,
1.5% corn gluten meal,
6.0% fish meal and
0.15% yeast extract; wherein the dextrin source is selected from corn dextrin (MS-3600) and maltodextrin.
PCT/US1997/001896 1996-02-07 1997-02-04 Effective production method of be-13793c WO1997029205A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388956A2 (en) * 1989-03-23 1990-09-26 Banyu Pharmaceutical Co., Ltd. Phthalinide derivative, pharmaceutical composition and use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388956A2 (en) * 1989-03-23 1990-09-26 Banyu Pharmaceutical Co., Ltd. Phthalinide derivative, pharmaceutical composition and use
US5290698A (en) * 1989-03-23 1994-03-01 Banyu Pharmaceutical Co., Ltd. Streptoverticillium ferm P-10489 which produces antitumor agent BE-13793C

Non-Patent Citations (4)

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Title
CHEMICAL ABSTRACTS, vol. 115, no. 13, 30 September 1991, Columbus, Ohio, US; abstract no. 131558, KOJIRI, KATSUHISA ET AL: "A new antitumor substance, BE-13793C, produced by a streptomycete. Taxonomy, fermentation, isolation, structure determination and biological activity" XP002032972 *
CHEMICAL ABSTRACTS, vol. 120, no. 11, 14 March 1994, Columbus, Ohio, US; abstract no. 124152, YAMADA, A. ET AL: "Structure and activity of new antibiotics exhibiting DNA topoisomerase inhibition" XP002032971 *
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TENNEN YUKI KAGOBUTSU TORONKAI KOEN YOSHISHU (1992), 34TH 259-266 CODEN: TYKYDS, 1992 *

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