Gene encoding glycosyltransferase and its uses

(1)

Preparation of chromosomal DNA from a strain of Saccharothrix aerocolonigenes and its cleavage with restriction enzymes

Saccharothrix aerocolonigenes ATCC39243 strain which had been grown by slant culture on a starch-inorganic salt agar medium (ISP4; manufactured by Difco) was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of Tryptocase Soy Broth (hereinafter abbreviated as TSB) medium (manufactured by Becton Dickinson Co.), and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days. Then, 2 ml of this culture medium was inoculated into TSB medium having 0.8% glycine added thereto, and incubated in the same manner for 2 days. 20 ml of the aforesaid culture medium was centrifuged to collect bacterial cells. These bacterial cells were centrifugally washed twice with TEG buffer [50 mM Tris-HCl buffer (pH 8.0), 10 mM EDTA.2Na, 50 mM glucose]. After the washed bacterial cells were resuspended in 10 ml of TEG buffer, 5 ml of a solution of lysozyme (manufactured by Sigma Co.; dissolved in TEG buffer at a concentration of 12 mg/ml) was added thereto and the resulting mixture was reacted at 37° C. for 1 hour. After 2 ml of 10% sodium dodecyl sulfate was added to lyse the bacterial cells, the mixture was alternately treated with phenol/chloroform (1:1) saturated with TE buffer [10 mM Tris-HCl pH 8.0), 1 mM EDTA-2Na], and chloroform/isoamyl alcohol (24:1). The aqueous layer was separated, 1/10 volume of 1 mg/ml ribonuclease A (RNase A; manufactured by Sigma Co.) was added thereto, and the resulting solution was incubated at 37° C. for 1 hour. After this solution was treated with an equal volume of chloroform/isoamyl alcohol (24:1), the aqueous layer was transferred to an autoclaved beaker and 1/10 volume of 3.3M sodium acetate was added thereto. While two volumes of cold ethanol was being added thereto, the solution was stirred with a glass rod to collect DNA thereon. The collected DNA was washed with 75% ethanol, dried and then dissolved in 9 ml of 0.1-fold SSC buffer [0.15M sodium chloride, 0.015M sodium citrate (pH 7.0)]. To the resulting DNA solution was added 1/10 volume of 3.3M sodium acetate. While 0.54 volume of isopropanol was being added thereto, the solution was stirred with a glass rod to collect DNA thereon. The collected DNA was washed with 75% ethanol, dried and then dissolved in an appropriate amount of TE buffer.

Next, 20 units of the restriction enzyme Sau3AI (manufactured by Takara Shuzo Co., Ltd.) was added to 50 μg of the above chromosomal DNA, and they were reacted in H buffer [50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothreitol, 100 mM sodium chloride] at 37° C. for 1 minute. All of the restriction enzymes used hereafter were ones manufactured by Takara Shuzo Co., Ltd. After completion of the reaction, the restriction enzyme was inactivated by treatment with phenol and extraction with ether. Then, the resulting DNA fragments were subjected to electrophoresis in 1.0% agarose/100 mM Tris-boric acid/2 mM EDTA.2Na (pH 8.3), and a DNA fraction having a size of about 2-10 Kbp was extracted with GENECLEAN II (manufactured by BIO 101 Co.).

(2)

Insertion of chromosomal DNA fragments into plasmid vector DNA

Streptomyces lividans TK21 strain, which is an actinomycete having the actinomycetal plasmid vector pIJ702, was inoculated into four 500 ml Erlenmeyer flasks containing 110 ml of the aforesaid TSB medium having 25 μg/ml of thiostrepton added thereto, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days. Bacterial cells were collected from 440 ml of this culture medium and centrifugally washed with the aforesaid TEG buffer, 50 ml of TEG buffer containing 2 mg/ml of lysozyme was added to the washed bacterial cells, and this suspension was allowed to stand at 37° C. for 30 minutes. After the addition of 50 ml of 0.2N NaOH/1% sodium dodecyl sulfate (SDS), the resulting mixture was allowed to stand at 0° C. for 5 minutes. Then, after the addition of 50 ml of 2.55M potassium acetate (pH 4.8), the resulting mixture was allowed to stand at 0° C. for 10 minutes and centrifuged. After the supernatant was treated with phenol, 3/5 volume of a polyethylene glycol 6000 (PEG-6000)/2.5M sodium chloride solution was added to the aqueous layer, and the resulting mixture was allowed to stand at 0° C. for 1 hour and centrifuged to precipitate DNA. This precipitate was dissolved in 4 ml of the aforesaid TE buffer and subjected to ethidium bromide-cesium equilibrium density gradient centrifugation to obtain plasmid DNA. The pIJ702 plasmid thus obtained was digested with the restriction enzyme BglII and incubated with 2 units of bacterial alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.) at 65° C. for 30 minutes. Then, the alkaline phosphatase was removed by treatment with phenol and extraction with ether. 1.0 μg of the phosphatase-treated plasmid DNA was mixed with 5 μg of the chromosomal DNA fragments obtained in (1), and they were ligated by means of a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.).

(3)

Transformation of Streptomyces lividans TK21 strain

The Transformation of Streptomyces lividans TK21 strain was carried out according to a modification of the method of D. A. Hopwood et al. (Genetic Manipulation of Streptomyces—a Laboratory Manual). Specifically, Streptomyces lividans TK21 strain which had been grown by slant culture on a yeast-malt agar medium (ISP2; manufactured by Difco) was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of the aforesaid TSB medium, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days. Then, 2 ml of this culture medium was inoculated into TSB medium having 0.5% glycine added thereto, and incubated in the same manner for 20 hours. Bacterial cells were collected from 40 ml of this culture medium and centrifugally washed twice with 0.35M P3 medium [11.98% sucrose, 0.4% sodium chloride, 0.1% magnesium chloride hexahydrate, 0.074% calcium chloride dihydrate, 0.573% N-tris(hydroxymethyl)methylaminoethane-sulfonic acid buffer (TES; manufactured by Dotite Co.; pH 7.2)]. Then, 20 ml of 0.35M PWP medium [11.98% sucrose, 0.4% sodium chloride, 0.2% magnesium chloride hexahydrate, 0.294% calcium chloride dihydrate, 0.573% TES buffer (pH 7.2)] was added to the washed bacterial cells, followed by intimate mixing. Thereafter, lysozyme was added thereto so as to give a final concentration of 10 mg/ml. This suspension was incubated at 30° C. for 60 minutes, passed through sterilized absorbent cotton to remove hyphae, and centrifuged. The precipitate was centrifugally washed twice with 0.35M PWP medium and suspended in 0.35M PWP medium so as to give a density of 10 9 to 10 10 protoplasts/ml. After a solution of the plasmid vector having DNA ligated thereinto was added to 100 μl of the protoplast suspension, 0.5 ml of 0.35M PWP medium having 30% PEG-2000 dissolved therein was further added thereto and mixed therewith. After 1 minute, 5 ml of 0.35M PWP medium was added thereto and protoplasts were collected by centrifugation. Then, these protoplasts were suspended in an appropriate amount of 0.35M PWP medium. 100 μl each of this suspension was applied onto 0.35M R3 agar plates [11.98% sucrose, 0.81% magnesium chloride hexahydrate, 0.05% potassium chloride, 0.22% calcium chloride dihydrate, 0.02% potassium dihydrogen phosphate, 1% glucose, 0.4% polypeptone, 0.04% yeast extract (manufactured by Difco), 0.573% TES buffer (pH 7.2), 2.2% agar], and incubated at 28° C. overnight. Then, 2.5 ml each of a melanin-producing soft agar medium (0.2% glucose, 1.0% yeast extract, 0.05% L-tyrosine, 0.5% sodium chloride, 0.4% agar) containing 250 μg/ml of thiostrepton was layered over these 0.35M R3 agar plates. Thereafter, the incubation was continued at 28° C. for an additional one week.

(4)

Detection of the desired gene

About 5,000 thiostrepton-resistant, non-melanin-productive strains obtained in the manner described in (3) were examined for glycosyltransferase activity. The activity was measured in terms of the activity for converting BE-13973C used as the substrate to ED-110. Specifically, each transformed strain was inoculated into a test tube containing 5 ml of a glycosylating medium [0.5% glucose, 8.0% dextrin, 0.25% L-threonine, 0.3% yeast extract, 0.1% magnesium sulfate heptahydrate, 0.2% calcium carbonate, 0.05% calcium chloride, 0.0002% ferrous sulfate, 0.00004% cupric chloride, 0.00004% manganese chloride, 0.00004% cobalt chloride, 0.00008% zinc sulfate, 0.00008% sodium borate, 0.00024% ammonium molybdate (pH 7.2)] to which thiostrepton had been added so as to give a final concentration of 20 μg/ml, and incubated at 28° C. on a rotary shaker (300 revolutions per minute) for 3 days. Thereafter, BE-13793C dissolved in methanol/tetra-hydrofuran (2:1) was added thereto so as to give a final concentration of 40 μg/ml, and the incubation was continued at 28° C. in the same manner for 2 days. The resulting culture medium was extracted with 5 ml of methyl ethyl ketone. After the methyl ethyl ketone layer was concentrated to dryness under reduced pressure, the resulting residue was dissolved in a small amount of tetrahydrofuran and subjected to thin-layer chromatography (TLC). Silica gel (Kieselgel 60; manufactured by Merck) was used on the thin-layer plate, and chloroform/methanol/tetrahydrofuran (3:1:1) was used as the developing solvent. In this case, the Rf value is 0.73 for BE-13793C and 0.36 for ED-110, so that they can be distinguished on the thin layer. When about 5,000 transformed strains were examined in this manner, one of them was found to have glycosyltransferase activity. Since this transformed strain has the plasmid pNGT206 as will be described later, it is conveniently designated as Streptomyces lividans TK21 (pNGT206). The plasmid pNGT206 contains a gene encoding glycosyltransferase.

(5)

Separation and analysis of the recombinant plasmid

According to the procedure described in (2), the DNA of the plasmid pNGT206 was purified from Streptomyces lividans TK21 (pNGT206) strain obtained in (4). The resulting plasmid was cleaved with various restriction enzymes to construct a restriction enyzme cleavage map. The results thus obtained are shown in FIG. 1 . The length of the inserted DNA fragment was 4.15 Kbp.

(6)

Preparation of pNGT207 from pNGT206

The plasmid pNGT206 obtained in (5) was reacted with the restriction enzyme PstI and subjected to electrophoresis in 1.0% agarose. Then, a 7.95 Kbp DNA fragment was extracted with GENECLEAN II. Using a DNA ligation kit, the extracted DNA fragment was ligated into the plasmid vector pIJ702. Then, according to the procedure described in (3), Streptomyces lividans TK21 strain was transformed with this vector to obtain a thiostrepton-resistant transformed strain. According to the procedure described in (4), it was confirmed that this transformed strain had glycosyltransferase activity. The strain obtained in the above-described manner was named Streptomyces lividans TK21 (pNGT207) strain and deposited with the Research Institute of Biotechnology and Industrial Technology, the Agency of Industrial Science and Technology (under the accession number of FERM P-14365). This strain was transferred on May 9, 1995 to international deposition based on the Budapest Treaty, and its accession number is FERM BP-5091. The restriction enzyme cleavage map of the plasmid pNGT207 is shown in FIG. 1 . The length of the inserted DNA fragment was 2.85 Kbp.

(7)

Preparation of a subcloned plasmid from the plasmid pNGT207 and determination of its base sequence

First of all, the plasmid pNGT207 was reacted with the restriction enzyme XhoI and subjected to electrophoresis in agarose. Then, a 1.9 Kbp DNA fragment was extracted with GENECLEAN II. Using a DNA ligation kit, the extracted DNA fragment was mixed with and ligated into the Escherichia coli plasmid vector pUC119 (manufactured by Takara Shuzo Co., Ltd.) which had been digested with SalI. After ligation, Escherichia coli MV1184 strain was transformed with this recombinant plasmid. The transformation of Escherichia coli MV1184 strain was carried out according to the following procedure. First of all, Escherichia coli MV1184 strain was inoculated into 100 ml of SOB medium [2% trypton (manufactured by Difco), 0.5% yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate), and incubated with shaking at 37° C. overnight. Then, 0.6 ml of this culture medium was inoculated into the same SOB medium and incubated at 37° C. As soon as the culture medium reached an absorbance of 11 at 550 mm, 50 ml of the culture medium was centrifuged to collect bacterial cells. These bacterial cells were suspended in 16 ml of TFB solution (10 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.3). 100 mM potassium chloride, 45 mM manganese chloride, 10 mM calcium chloride, 3 mM cobalt chloride] and allowed to stand at 0° C. for 10 minutes. Thereafter, the bacterial cells were collected by centrifugation and resuspended in 3.8 ml of TFB solution. After the solution of 133 μl of dimethyl sulfoxide, the suspension was allowed to stand at 0° C. for 15 munites. Thereafter, 133 μl of dimethyl sulfoxide was added thereto and the suspension was allowed to stand at 0° C. for 5 munites. The mixture having undergone the above-described DNA ligation was added to 100 μl of this suspension, and the resulting mixture was allowed to stand at 0° C. for 1 hour. After this mixture was heat-treated at 42° C. for 45 seconds, 400 μl of SOB medium was added thereto and the resulting mixture was incubated with shaking at 37° C. for 1 hour. Thereafter, 100 μl each of the mixture was applied to X-Gal plates (1.0% trypton, 0.25% sodium chloride, 1.5% agar) containing ampicillin (100 μg/ml). 5-bromo-4-chloro-3-indolyl-β-D-gal actoside (X-gal; 40 μg/ml) and isopropyl-β-D-thiogalactopyranoside (23.8 μg/ml), and incubated statically at 37° C. overnight. Then, a transformed strain of Escherichia coli forming an ampicillin-resistant white colony was selected, and the plasmid were extracted and purified from this transformed strain. The purification of the recombinant plasmid was carried out according to the procedure described in (2). However, when the plasmid DNA of Escherichia coli was to be purified, the transformed strain was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of twofold YT medium [0.8% bactotrypton, 0.5% yeast extract, 0.5% sodium chloride (pH 7.6)] containing 100 μg/ml of ampicillin, and incubated at 37° C. on a rotary shaker (200 revolutions per minute) overnight.

The 0.9 Kbp XhoI fragment of pNGT207 shown in FIG. 1 encodes a protein having glycosyltransferase activity. Using the Escherichia coli plasmid vectors pUC118 (manufactured by Takara Shuzo Co., Ltd.) and pUC119, its base sequence was determined by means of a base sequence determination kit (BcaBEST™ Dideoxy Sequencing Kit; manufactured by Takara Shuzo Co., Ltd.).

(8)

Analysis of the structure of the glycosyltransferase gene

The base sequence data thus obtained were analyzed by means of the DNA analysis program GCG (a program made by Genetic Computer Group Co.). The base sequence so determined consists of 1,898 base pairs, has an open reading frame of 1,245 base pairs extending from the 285th base (ATG) to the 1529th base (TGA), and encodes a protein composed of 414 amino acids residues.

(9)

Transformation of Streptomyces mobaraensis BA 13793 strain with the plasmid pNGT207

Streptomyces mobaraensis BA13793 strain which had been grown by slant culture on ISP4 medium containing 25 μg/ml of thiostrepton was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of TSB medium, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days. Moreover, 2 ml of this culture medium was inoculated into TSB medium having 0.8% glycine added thereto, and incubated under the same conditions as described above for 2 days. 40 ml of this culture medium was centrifuged to collect bacterial cells, which were centrifugally washed twice with sterilized 0.5M P3 medium [17.1% sucrose, 0.4% sodium chloride, 0.1% magnesium chloride hexahydrate, 0.074% calcium chloride di hydrate, 0.573% TES buffer (pH 7.2)]. Then, 20 ml of sterilized 0.5M PWP medium [17.1% sucrose, 0.4% sodium chloride, 0.2% magnesium chloride hexahydrate, 0.294% calcium chloride dihydrate, 0.573% TES buffer (pH 7.2)] was added to the washed bacterial cells, followed by intimate mixing. Thereafter, lysozyme was added thereto so as to give a final concentration of 750 μg/ml, and Achromopeptidase (manufactured by Wako Pure Chemical Industries Ltd.) was added thereto so as to give a final concentration of 500 μg/ml. This suspension was incubated at 37° C. for 1 hour, passed through sterilized absorbent cotton to remove hyphae, and centrifuged. The precipitate was centrifugally washed twice with 0.5M PWP medium and suspended in 0.5M PWP medium so as to give a density of 10 9 to 10 10 protoplasts/ml. After an appropriate amount of the plasmid pNGT207 was added to 100 μl of the protoplast suspension, 0.5 ml of 0.5M PWP medium having 30% PEG-2000 dissolved therein was further added thereto and mixed therewith. After 1 minute, 5 ml of 0.5M PWP medium was added thereto and the protoplasts were centrifugally washed. Then, these protoplasts were suspended in an appropriate amount of 0.5M PWP medium, 100 μl each of this suspension was applied onto 0.5M R3 agar plates [17.1% sucrose, 0.81% magnesium chloride hexahydrate, 0.05% potassium chloride, 0.22% calcium chloride dihydrate, 0.02% potassium dihydrogen phosphate, 1% glucose, 0.4% polypeptone, 0.04% yeast extract, 0.573% TES buffer (pH 7.2), 1.0% gellan gum], and incubated at 37° C. Then, 2.5 ml each of a melanin-producing soft agar medium [comprising 0.5M R3 agar medium in which the gellan gum is replaced by 0.4% low-melting agarose (manufacturing by Nakarai Co., Ltd.)] was layered over these agar plates. Thereafter, the incubation was continued at 28° C. for an additional two weeks.

The resulting transformed strain, Streptomyces mobaraensis BA13793 (pNGT207), was deposited with the Research Institute of Biotechnology and Industrial Technology, the Agency of Industrial Science and Technology (under the accession number of FERM P-14364). This strain was transferred on May 9, 1995 to international deposition based on the Budapest Treaty, and its accession number is FERM BP-5090.

Procedure for the preparation of ED-110 by use of the transformed strain Streptomyces lividans TK21 (pNGT207)

Streptomyces lividans TK21 (pNGT207) strain which had been grown by slant culture on ISP2 medium containing 25 μg/ml of thiostrepton was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of a culture medium (pH 7.2) composed of 2.0% glucose, 4% soluble starch, 0.3% yeast extract, 0.5% polypeptone, 1.0% corn gluten meal, 0.2% ammonium sulfate, 0.2% sodium chloride and 0.2% calcium carbonate, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 2 days. Then, 1 ml each of this culture medium was inoculated into two 500 ml Erlenmeyer flasks containing 110 ml of a culture medium (pH 7.2) composed of 2.0% glucose, 6.0% soluble starch, 0.3% yeast extract, 0.5% polypeptone, 1.0% soya flour (manufactured by Nisshin Flour Milling Co., Ltd.), 0.2% sodium chloride, 0.2% ammonium sulfate, 0.2% calcium carbonate and 0.01% ferrous sulfate heptahydrate, and incubated under the same conditions as described above. After two days of incubation, 1 ml each of a methanol/tetrahydrofuran (1:1) mixture containing 120 mg of BE-13793C was added to the flasks, and the incubation was continued under the same conditions as described above for an additional 7 days.

The bacterial cells obtained by filtering the aforesaid culture media were extracted twice with 200 ml portions of tetrahydrofuran. After these extracts were combined and concentrated to dryness, 200 ml of water was added thereto. This mixture was extracted twice with 200 ml portions of ethyl acetate. After the resulting ethyl acetate layers were combined and concentrated to dryness, the resulting residue was subjected to column chromatography (2.0×40 cm) using silica gel (Kieselgel 60; manufactured by Merck), and developed with chloroform/methanol/tetrahydrofuran (4:1:1) to obtain fractions containing the desired compound. After these fractions were combined and concentrated to dryness, the resulting residue was dissolved in a small amount of methanol, subjected to column chromatography (1.5×60 cm) using Sephadex LH20 (manufactured by Pharmacia Co.), and eluted with methanol to obtain fractions containing the desired compound alone. These fractions were combined and concentrated to dryness to obtain 12.0 mg of the title compound.

Rf value: 0.51 [column, Kieselgel 60F 254 (manufactured by Merck); developing solvent, chloroform/methanol/tetrahydrofuran (2:1:1)].

HPLC: Rt 6.18 minutes [column, Chromatorex ODS having an inner diameter of 4.6 mm and a length of 250 mm; detection, UV 305 nm; flow rate, 1.0 ml/min.; mobile phase, methanol/water (3:1)].

FAB-MS (m/z): 519 [M] +

1 H-NMR (400 MHz, DMSO-d 6 , δ ppm): 11.0(1H, s), 10.9(1H, s), 10.3(1H, brs), 9.93(1H, brs), 8.69(1H, d, J=7.8 Hz), 8.51(1H, d, J=7.8 Hz), 7.17(2H, t, J=7.8 Hz), 7.05(1H, d, J=9.3 Hz), 7.01(1H, d, J=7.8 Hz), 6.99(1H, d, J=7.8 Hz), 5.41(1H, d, J=5.9 Hz), 5.34(1H, brs), 5.20(1H, d, J=5.4 Hz), 4.89(1H, brs), 4.02(2H, m), 3.74(1H, m), 3.63(2H, m), 3.41(1H, m).

Procedure for the preparation of the compound of the formula

by use of Streptomyces lividans TK-21 (pNGT207) strain

Streptomyces lividans TK21 (pNGT207) strain which had been grown on ISP2 slant agar medium containing 20 μg/ml of thiostrepton was inoculated into two 500 ml Erlenmeyer flasks containing 110 ml of a culture medium (pH 7.2 before sterilization) composed of 8.0% MS3600, 0.5% glucose, 0.25% L-threonine, 0.3% yeast extract, 0.1% magnesium sulfate hydrate, 0.2% calcium carbonate, 0.0002% ferrous sulfate, 0.00004% cupric chloride, 0.00004% manganese chloride, 0.00004% cobalt chloride, 0.0008% zinc sulfate, 0.00008% sodium borate, 0.00024% ammonium molybdate and 0.5% 3-(N-morpholino)propanesulfonic acid, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days. Then, 1 ml each of this culture medium was inoculated into one hundred 500 ml Erlenmeyer flasks containing 110 ml of a culture medium composed of the same components as described above, and incubated at 28° C. on a rotary shaker (180 revolutions per minute). After three days of incubation, 1 ml each of a methanol/tetrahydrofuran (3:1) mixture containing 10 mg of the compound of formula [VI] (Reference Example 1) was added to the flasks, and the incubation was continued under the same conditions as described above for an additional 11 days.

The bacterial cells obtained by filtering the aforesaid culture media were extracted twice with 5 L portions of methanol. After these extracts were combined and concentrated to remove the methanol, 1 L of water was added thereto. This mixture was successively extracted with 1.5 L, 1 L and 0.5 L of ethyl acetate. After the resulting ethyl acetate layers were combined and concentrated to dryness, the resulting residue was subjected to column chromatography (2.5×40 cm) using silica gel (Kieselgel 60; manufactured by Merck). The residue was washed with 1.5 L of chloroform/methanol (5:1) and eluted with a similar solvent system (3:1) to obtain fractions containing the desired compound. After these fractions were combined and concentrated to dryness, the resulting residue was dissolved in a small amount of ethanol, subjected to column chromatography (3×60 cm) using Sephadex LH20 (manufactured by Pharmacia Co.), and developed with ethanol to obtain fractions containing the desi red compound. These fractions were combined and concentrated to dryness to obtain 250 mg of the title compound.

Rf value: 0.19 [column, Kieselgel 60F 254 (manufactured by Merck); developing solvent, chloroform/methanol/tetrahydrofuran (3:1:1)].

HPLC: Rt 20.0 minutes (column, Chromatorex ODS having an inner diameter of 4.6 mm and a length of 250 mm; detection. UV 305 nm; flow rate, 1 ml/min.; mobile phase, a linear gradient from 50% to 100% methanol over a period of 30 minutes).

FAB-MS (m/z): 534 [M+H] +

1 H-NMR (400 MHz, DMSO-d 6 , δ ppm): 11.2(1H, s), 9.76(2H, brs), 8.88(1H, d, J=8.3 Hz), 8.80(1H, d, J=8.8 Hz), 7.18(1H, d, J=2.0 Hz), 6.98(1H, d, J=2.0 Hz), 6.83(1H, dd, J=8.3, 2.0 Hz), 6.80(1H, dd, J=8.8, 2.0 Hz), 5.98(1H, d, J=8.8 Hz), 5.87(1H, t, J=3.4 Hz), 5.35(1H, d, J=4.9 Hz), 5.13(1H, d, J=3.9 Hz), 4.95(1H, d, J=4.4 Hz), 4.01(1H, dd, J=10.7, 3.4 Hz), 3.91(2H, m), 3.77(1H, m), 5 3.51(2H, m), 3.15(3H, s).

Procedure for the preparation of the compound of the formula

by use of Streptomyces lividans TK-21 (pNGT207) strain

Streptomyces lividans TK21 (pNGT207) strain which had been grown by slant culture on ISP2 medium containing 25 μg/ml of thiostrepton was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of a culture medium (pH 7.2) composed of 2.0% glucose, 4% soluble starch, 0.3% yeast extract, 0.5% polypeptone, 1.0% corn gluten meal, 0.2% ammonium sulfate, 0.2% sodium chloride and 0.2% calcium carbonate, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 2 days. Then, 1 ml each of this culture medium was inoculated into ten 500 ml Erlenmeyer flasks containing 110 ml of a culture medium (pH 7.2) composed of 2.0% glucose, 6.0% soluble starch, 0.3% yeast extract, 0.5% polypeptone, 1.0% soya flour (manufactured by Nisshin Flour Milling Co., Ltd.), 0.2% sodium chloride, 0.2% ammonium sulfate, 0.2% calcium carbonate and 0.01% ferrous sulfate heptahydrate, and incubated under the same conditions. After two days of incubation, 0.5 ml each of a dimethyl sulfoxide solution containing 10 mg of the compound of formula [VII] (Reference Example 2) was added to the flasks, and the incubation was continued under the same conditions as described above for an additional 7 days.

The bacterial cells obtained by filtering the aforesaid culture media were extracted with 500 ml of tetrahydrofuran. After this extract was concentrated to dryness, 200 ml of water was added thereto. This mixture was extracted twice with 200 ml portions of ethyl acetate. After the resulting ethyl acetate layers were combined and concentrated to dryness, the resulting residue was subjected to column chromatography (2.0×35 cm) using silica gel (Kieselgel 60; manufactured by Merck).

The residue was washed with 700 ml of chloroform/methanol/tetrahydrofuran/25% aqueous ammonia (20:10:10:1) and then 250 ml of a similar solvent system (20:40:10:1), and eluted with a similar solvent system (30:60:5:2) to obtain fractions containing the desired compound. After these fractions were combined and concentrated to dryness, the resulting residue was dissolved in a small amount of a methanol/tetrahydrofuran (1:1) mixture, subjected to column chromatography (1.5×25 cm) using Sephadex LH20 (manufactured by Pharmacia Co.), and developed with methanol/tetrahydrofuran (4:1). The resulting eluate was concentrated to dryness to obtain 17.6 mg of the title compound as a crude product. Moreover, 10 mg of this crude product was dissolved in 2.5 ml of tetrahydrofuran, After the addition of 5 ml of methanol, the resulting mixture was allowed to stand at 4° C. for 15 hours. Thus, 4.3 mg of the title compound was obtained.

RF value: 0.28 [column, Kieselgel 60F 254 (manufactured by Merck); developing solvent, chloroform/methanol/tetrahydrofuran (3:1:1)].

HPLC: Rt 17.5 minutes (column, Chromatorex ODS having an inner diameter of 4.6 mm and a length of 250 mm; detection, UV 305 nm; flow rate, 1 ml/min.; mobile phase, a linear gradient from 50% to 100% methanol over a period of 30 minutes).

FAB-MS (m/z): 535 [M+H] +

1 H-NMR (400 MHz, DMSO-d 6 , δ ppm): 10.9(1H, s), 10.4(1H, s), 10.0(1H, s), 8.73(1H, d, J=7.8 Hz), 8.55(1H, d, J=7.8 Hz), 7.19(2H, t, J=7.8 Hz), 7.05(1H, d, J=9.3 Hz), 7.02(1H, d, J=7.8 Hz), 6.90(1H, d, J=7.8 Hz), 5.42(1H, d, J=5.9 Hz), 5.34(1H, brs), 5.22(1H, brs), 4.96(2H, brs), 4.91(1H, d, J=4.9Hz), 4.01(2H, m), 3.74(1H, m), 3.63(2H, m), 3.39(1H, m).

Procedure for the preparation of ED-110 by use of the transformed Streptomyces mobaraensis BA13793 (pNGT207) strain

The transformed Streptomyces mobaraensis BA13793 (pNGT207) strain which had been grown by slant culture was inoculated into a 500 ml Erlenmeyer flask containing 110 ml of a culture medium (pH 7.2 before sterilization) composed of 0.1% glucose, 2.0% dextrin, 1.0% corn gluten meal, 0.5% fish meal, 0.1% yeast extract, 0.1% sodium chloride, 0.05% magnesium sulfate, 0.05% calcium chloride, 0.0002% ferrous sulfate, 0.00004% cupric chloride, 0.00004% manganese chloride, 0.00004% cobalt chloride, 0.00008% zinc sulfate, 0.00008% sodium borate, 0.00024% ammonium molybdate and 0.5% 3-(N-morpholino)propanesulfonic acid, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 3 days.

Then, 2 ml each of this culture medium was inoculated into three 500 ml Erlenmeyer flasks containing 110 ml of a culture medium (pH 7.2 before sterilization) composed of 0.2% glucose, 4.0% dextrin, 1.5% corn gluten meal, 0.15% yeast extract, 3.0% fish meal, 0.1% sodium chloride, 0.05% magnesium sulfate, 0.05% calcium chloride, 0.0002% ferrous sulfate, 0.00004% cupric chloride, 0.00004% manganese chloride, 0.00004% cobalt chloride, 0.00008% zinc sulfate, 0.00008% sodium borate, 0.00024% ammonium molybdate and 0.5% 3-(N-morpholino)propanesulfonic acid, and incubated at 28° C. on a rotary shaker (180 revolutions per minute) for 17 days.

The bacterial cells obtained by filtering the aforesaid culture media were extracted twice with 500 ml portions of tetrahydrofuran. After these extracts were combined and concentrated to dryness, 300 ml of water was added thereto. This mixture was extracted twice with 300 ml portions of ethyl acetate. After the resulting ethyl acetate layers were combined and concentrated to dryness, the resulting residue was subjected to column chromatography (2.0×40 cm) using silica gel (Kieselgel 60; manufactured by Merck), and developed with chloroform/methanol/tetrahydrofuran (4:1:1) to obtain fractions containing the desired compound. After these fractions were combined and concentrated to dryness, the resulting residue was dissolved in a small amount of a methanol/tetrahydrofuran mixture and subjected to column chromatography (φ1.5×60 cm) using Sephadex LH20 (manufactured by Pharmacia Co.).

The residue was developed with methanol/tetrahydrofuran (5:1). After fractions containing the desired compound (ED-110) were combined and concentrated to dryness, the resulting residue was subjected to column chromatography (1.5×60 cm) using Sephadex LH20 (manufactured by Pharmacia Co.), and developed with ethanol. The resulting eluate was concentrated to dryness to obtain 68.3 mg of ED-110. In the aforesaid silica gel chromatography, 287.5 mg of BE-13793C was obtained by concentrating BE-13793C-containing fractions to dryness. The property data of ED-110 thus obtained were identical those of ED-110 obtained in Example 2 by use of Streptomyces lividans TK-21 (pNGT207) strain.

Reference Example 1

Procedure for the preparation of the compound of the formula

a) A tetrahydrofuran solution (0.93M, 6.9 ml) containing ethylmagnesium bromide was warmed to 45° C. and a toluene solution (7.8 ml) containing 6-methoxyindole (949 mg) was added thereto. After 40 minutes, a toluene solution (7.8 ml) containing 2,3-dibromo-N-methylmaleimide (388 mg) was added dropwise thereto over a period of 40 minutes, and the resulting mixture was refluxed for 2 hours. After the reaction mixture was cooled with ice, a 20% aqueous solution of citric acid was added thereto. This mixture was extracted with ethyl acetate, and the organic solvent layer was concentrated under reduced pressure. The resulting residue was subjected to Sephadex LH-20 column chromatography and developed with methanol to obtain 415 mg of 2,3-bis(6-methoxy-1H-indol-3-yl)-N-methylmaleimide (in a 72% yield).

b) 410 mg of the 2,3-bis(6-methoxy-1H-indol-3-yl)-N-methylmaleimide obtained in Reference Example 1-a). 255 mg of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and a catalyst amount of p-toluene sulfonic acid were added to 150 ml of dry benzene, and this mixture was refluxed in a stream of argon for 45 minutes. After the reaction mixture was cooled, a saturated aqueous solution of sodium thiosulfate was added thereto. This mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate and then a saturated aqueous solution of sodium chloride, and concentrated under reduced pressure. The resulting residue as subjected to silica gel chromatography (chloroform/methanol/tetrahydrofuran=30:1:1) and developed with chloroform/methanol (10:1) to obtain 250 mg of trimethylarcyriaflavin C (in a 62% yield).

c) A mixture of 250 mg of the trimethylarcyriaflavin C obtained in Reference Example 1-b) and 2.2 g of pyridine hydrochloride was heated at 180° C. n a sealed tube for 90 minutes. The reaction mixture was cooled, diluted with N,N-dimethylformamide and 1N hydrochloric acid, and then extracted with ethyl acetate. The organic layer was washed with water and then a saturated aqueous solution of sodium chloride, and concentrated to dryness under reduced pressure. The resulting residue was subjected to Sephadex LH-20 column chromatography and developed with methanol to obtain 131 mg of the title compound of formula [VI] (6-methylarcyriaflavin C) (in a 56% yield).

FAB-MS (m/z): 372 [M+H] +

1 H-NMR (300 MHz, DMSO-d 6 , δppm): 11.35(2H, s), 9.73(2H, s), 8.70(2H, d, J=8.6 Hz), 7.04(2H, d, J=2.0 Hz), 6.78(2H, dd, J=8.6, 2.0 Hz), 3.14(3H, s).

Reference Example 2

Procedure for the preparation of the compound of the formula

0.52 of BE-13793C was dissolved in 3 ml of hydrazine hydrate (manufactured by Wako Pure Chemical Industries Ltd.), and this solution was stirred at room temperature for 1 hour. Then, concentrated hydrochloric acid was added thereto until the solution became acidic. The resulting precipitate was collected by filtration, washed with purified water, and dried under reduced pressure to obtain 0.53 g of the title compound of formula [VII] (in a 98% yield).

Rf value: 0.63 [column, Kieselgel 60F 254 (manufactured by Merck); developing solvent, chloroform/methanol/tetrahydrofuran (3:1:1)].

HPLC: 19.1 minutes (column, Chromatorex ODS having an inner diameter of 4.6 mm and a length of 250 mm; detection, UV 305 nm; flow rate, 1 ml/min.; mobile phase, a linear gradient from 50% to 100% methanol over a period of 30 minutes).

FAB-MS (m/z): 373 [M+H] +

1 H-NMR (400 MHz, DMSO-d 6 , δ ppm): 11.6(2H, s), 10.2(2H, s), 8.44(2H, d, J=7.8 Hz), 7.14(2H, t, J=7.8 Hz), 6.98(2H, d, J=7.8 Hz), 4.90(2H, s).