JP2003174884A - rhoG GENE AND USE THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide DNAs encoding glycosyltransferases associated with biosynthesis of an anthracycline antibiotic, a method for producing the anthracycline antibiotic with a microorganism bred by using the DNAs and the new anthracycline antibiotic produced by the method. <P>SOLUTION: The DNA (corresponding to a gene of the glycosyltransferase having the ability to transfer rhodosamine) represented by base 914 to base 2191 of a base sequence having a specific sequence of sequence number 1 (refer to the specification) can be obtained by a PCR method using a genomic DNA of Streptomyces violaceus A262, SC-7 strain as a template. The new anthracycline antibiotic is produced from a strain bred by using the DNA and the gene of the glycosyltransferase having the ability to transfer daunosamine and derived from the daunomycin-producing microorganism. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding a glycosyltransferase involved in biosynthesis of anthracycline antibiotics, and a method for producing anthracycline antibiotics by a microorganism bred using the DNA. And a novel anthracycline antibiotic produced by the method.

[0002]

2. Description of the Related Art Anthracycline antibiotics are pigment glycosides (glycosides) composed of an aglycone moiety having a four-membered ring quinone structure as a basic skeleton and sugars mainly composed of amino sugars. Although it has a broad spectrum cancer control spectrum,
It is a carcinostatic antibiotic that has difficulty in accumulating cardiotoxic action, and it is daunorubicin, doxorubicin.
(doxorubicin), aclarubicin (aclarubicin), sinervins (cinerubins), rhodomycins (rhodomycins),
Nogalamycins and stefimycins
Many substances such as (steffimycins) are known.

All of these antibiotics have been isolated from cultures of microorganisms belonging to actinomycetes, and a wide variety of derivatives have been obtained by mutant strains derived from these microorganisms. In addition to these methods, attempts have been made to produce new anthracycline antibiotics by using microorganisms obtained by genetically modifying them (for example, Micr
obiology 142,1965-1972 (1996), J. Antibiotics 49,355
-359 (1996), J. Antibiotics 53, 828-836 (2000), etc.).

As described above, various derivatives of anthracycline antibiotics as anticancer agents have been proposed.
Nothing is satisfactory in terms of both toxicity and carcinogenicity. Moreover, since the results of the in vitro test and the animal test cannot always be directly reflected as the carcinostatic effect on humans, the carcinostatic agent requires multifaceted research. Therefore, even with respect to the group of anthracycline antibiotics, which are tentatively evaluated as anticancer agents, it is desired to propose compounds belonging to a new category.

[0005]

The present inventors have introduced a gene recombination method into breeding of microorganisms having the ability to produce anthracycline antibiotics, in order to search for anthracycline antibiotics having excellent carcinostatic action. I considered to do. First, by using a primer designed with reference to a highly conserved region in the amino acid sequence of a known glycosyltransferase, a microorganism Streptomyces which has the ability to produce the anthracycline antibiotic rhodomycin
Genomic DN of Streptomyces violaceus A262, SC-7 (hereinafter sometimes abbreviated as "A262, SC-7 strain")
A gene encoding a glycosyltransferase having the ability to transfer rhodosamine from A to ε-rhodomycinone (hereinafter sometimes abbreviated as “rhodosamine transferase”) (hereinafter, “rho
A part of "G" may be abbreviated) by the polymerase chain reaction (hereinafter sometimes abbreviated as "PCR") method. A gene encoding a rhodosamine transferase was successfully isolated by screening a genomic DNA library of the microorganism using the obtained amplified fragment as a probe.

Next, using the thus obtained rhoG gene, a gene disruption vector containing DNA lacking rhodosamine transferability was constructed by utilizing the frame shift caused by blunting of the internal restriction enzyme site. Furthermore, by double homologous recombination (double crossover) using this vector,
A mutant strain (rhoG-disrupted strain) was prepared by introducing a mutation lacking rhodosamine transfer ability into the genomic DNA of A262, SC-7 strain.

On the other hand, the microorganism Streptomyces peucetius ATCC125 having the ability to produce the anthracycline antibiotic daunomycin
From 20 genomic DNA, a gene (hereinafter, abbreviated as "dnrS") encoding a glycosyltransferase having the ability to transfer daunosamine to ε-rhodomycinone (hereinafter sometimes abbreviated as "daunosamine transferase") Was obtained by amplification by the PCR method. The obtained dnrS gene was incorporated into a plasmid that was expressed in actinomycetes, and was introduced into the rhoG-disrupted strain prepared above. As a result, the transformants obtained had relatively excellent carcinostatic activity The present invention has been completed by finding that there are microorganisms that produce anthracycline antibiotics.

The present invention has been made based on these findings, and the present invention is a DN encoding a rhodosamine transferase involved in biosynthesis of anthracycline antibiotics.
A, introducing a gene encoding a heterologous glycosyltransferase into a mutant strain bred using the DNA and lacking rhodosamine transferability,
It is intended to provide a method for producing an anthracycline antibiotic using the expressed transformant and a novel anthracycline antibiotic having an excellent carcinostatic action.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION A DNA encoding a protein having the ability to transfer rhodosamine to ε-rhodomycinone of the present invention, or a modified form thereof, is represented by the following (a), (b) or (c): is there. (a) A DNA consisting of a continuous base sequence from base 914 to base 2191 of SEQ ID NO: 1. (b) a modified form of the DNA shown in (a) above, (i) above
(a) A DNA that hybridizes with the DNA shown under stringent conditions and (ii) encodes a protein having the ability to transfer rhodosamine to ε-rhodomycinone.
A. (c) Due to the degeneracy of gene codons, the DNA shown in (a) above
DNA that does not hybridize with the above under stringent conditions, but encodes a protein having the same amino acid sequence as the protein encoded by the DNA represented by (a) or (b) above.

The DNA of the present invention can be obtained from any actinomycete that has the ability to produce rhodomycin, and as a typical example, Streptomyces violaceus is used.
(Storeptomyces violaceus) A262, SC-7 can be mentioned. This strain was designated as FERM B by the Institute of Microbial Science and Technology (now AIST) on March 31, 1986.
Deposited as P-1331.

The method for obtaining the DNA of the present invention may be any method, but preferably, in the amino acid sequence of a glycosyltransferase derived from a microorganism producing an anthracycline antibiotic or a macrolide antibiotic,
Polymerase chain reaction using primers designed with reference to highly conserved regions (P
CR) method.

For example, the DNA encoding the rhodosamine transferase of the present invention can be obtained as follows. Under certain culture conditions, Streptomyces violaceus (Strep
tomyces violaceus) A262, SC-7 is cultivated, the obtained bacterial cells are crushed to obtain genomic DNA or mRNA, and a genomic DNA library or a cDNA library is prepared according to a conventional method. On the other hand, in the amino acid sequences of glycosyltransferases of microorganisms that produce anthracycline antibiotics or macrolide antibiotics, highly conserved regions (see, for example, J. Bacteriol. 177, 6688-6692 (1995)). ) To design and prepare primers. By using the prepared primers and performing a PCR reaction using the genomic DNA of the A262, SC-7 strain as a template, a part of the DNA encoding rhodosamine transferase can be amplified.

Using a primer designed based on a DNA fragment which is a part of the DNA coding for the rhodosamine transferase thus amplified or its nucleotide sequence as a probe,
By screening the above-mentioned genomic DNA library or cDNA library, a clone containing the DNA encoding the target rhodosamine transferase can be isolated, and the DNA encoding the rhodosamine transferase can be obtained from this clone.

Further, by using as a probe an oligonucleotide synthesized on the basis of the nucleotide sequence of the DNA encoding the rhodosamine transferase revealed by the present invention, Streptomyces violaceus (Strepto
It is also possible to screen a DNA library derived from a microorganism belonging to myces violaceus) A262, SC-7 or other actinomycetes, and obtain the DNA encoding the target rhodosamine transferase from the isolated clone.

Further, the DNA of the present invention is not only the above-mentioned DNA but also a modified form thereof, which is 2 × S at 60 ° C. under constant hybridization conditions with respect to the above-mentioned DNA.
0.5 × SSC in SC (standard citrate saline), preferably at 60 ℃
In particular, it also includes a variant that encodes a protein having a nucleotide sequence that hybridizes under stringent conditions in 0.2 × SSC at 60 ° C., and having the ability to transfer rhodosamine to ε-rhodomycinone. . More specific examples of such variants include the following.

The above-mentioned modified DNA which encodes a protein having an ability to transfer rhodosamine to ε-rhodomycinone is at least 55%, preferably 70%, more preferably 70% with the nucleotide sequence from base 914 to base 2191 in SEQ ID NO: 1. Indicates 95% homology. Such variants include those in which the bases have been removed or added at the 5'end or 3'end or in the middle of the base sequence, respectively, or those in which some of the bases have been replaced with other bases. . Variants in which a part of the bases are replaced with other bases include those encoding the same protein but having a base sequence different from that of the DNA due to the degeneracy of the genetic code.

For substitution of bases other than those associated with the degeneracy of the genetic code, reference should be made to the deduced amino acid sequences encoded by the respective DNAs, and the similarity of side chains derived from the respective amino acids, for example, hydrophobicity and hydrophilicity It is preferable that the protein as a whole has a similar shape based on the electric charge, the size, and the like. The variant is a DNA having a function equivalent to that of the DNA,
That is, it is estimated that a protein encoding a protein having the ability to transfer rhodosamine to ε-rhodomycinone can be obtained with a considerably high probability.

Such a modified form of the present invention is obtained by
Can be synthesized using a nucleic acid synthesizer, or can be prepared by point mutagenesis or site-directed mutagenesis known per se, with reference to the nucleotide sequences of E. coli or deduced amino acid sequences encoded by them.

The form of use of the DNA of the present invention is to construct a microorganism into which these DNAs are incorporated, express these DNAs, and produce a protein having the ability to transfer rhodosamine to its encoded ε-rhodomycinone. It is to let. The site for incorporating the DNA of the present invention or a variant thereof may be either on the plasmid of the host microorganism or on the chromosome. Such a plasmid can be autonomously replicating, including an autonomously replicating sequence, a promoter sequence, a terminator sequence, a drug resistance gene, etc., in addition to the above-mentioned DNA or variants thereof. Further, the plasmid can be an integrative plasmid having a sequence homologous to a certain region of the genome of the host to be used.

As described above, the host and plasmid vector system for expressing the protein encoded by the DNA of the present invention may be any system as long as these DNAs are stably retained and expressed. If the strain from which the gene encoding the rhodosamine transferase is derived or the actinomycete of its relatives is used as the host, the autonomously replicating vector pIJ6021 (Gene
166,133-137 (1995)) and chromosome integration type vector KC515
(The bacteria, vol.9.Antibiotic-producing Streptomy
ces (ed: Queener, SE and Day, LE) .p.119-158.Academ
ic Press, Orlando, Fgla.) etc. can be used.

[0021] Another use form of the DNA of the present invention is to construct a gene disruption vector containing a modified DNA in which the nucleotide sequence inside these DNAs is modified to lack the glycosyl transfer ability, and this vector is used. This is to prepare a rhoG-disrupted strain in which a mutation lacking the ability of glycosyl transfer is introduced into the genomic DNA of the host microorganism.

The modification of the nucleotide sequence for deleting these glycosyl transfer ability can be carried out by utilizing, for example, a frameshift mutation caused by blunting an appropriate restriction enzyme site inside DNA. Examples of suitable restriction enzymes include SacI and PvuII. The modified DNA thus obtained is then transformed into a suitable vector plasmid, for example p
By incorporating it into UC19 (Gene 33, 103-119 (1985)), a gene disruption vector containing a DNA encoding a protein lacking the ability to transfer rhodosamine is constructed. Then, this gene disruption vector is subjected to a conventional method, and an actinomycete capable of producing rhodomycin, for example, Streptomyces violaceus A262, SC-
A transformant strain integrated in 7 is obtained, and a strain that produces ε-rhodomycinone and has a plasmid-derived marker is selected from those that have grown into protoplasts, regenerated, and grown. In this way, DNA derived from the gene disruption vector was integrated into the host, and single cross
r) A strain can be obtained.

Then, the obtained single-homologous recombination strain is sporulated, protoplasted, and regenerated repeatedly to produce ε-rhodomycinone, and a double-homologous recombination in which a plasmid-derived marker other than the modified DNA is released ( d
ouble crossover) strain can be obtained. These rhoG
The frequency of acquisition of the disrupted strain is about 0.5 to several percent at both the single-time homologous recombination and the double-time homologous recombination stages, and can be easily acquired by those skilled in the art.

According to the present invention, the above-mentioned rhoG-disrupted strain as a host is transformed with a plasmid vector that has been recombined so as to contain a DNA encoding a protein having the ability to transfer sugars other than rhodosamine to ε-rhodomycinone. The transformant thus obtained is also provided. A specific example of such a transformant is Streptomyces, which is a daunomycin-producing bacterium.
peucetius) ATCC12520 strain-derived daunosamine transferase-encoding dnrS gene into a vector plasmid for actinomycetes such as pI6021 and transformed with a recombinant vector.

Furthermore, according to the present invention, the following formula (I) using the above transformant is used.

[Chemical 4] (However, in the formula, R ₁ is a hydrogen atom or

[Chemical 5] In which R ₂ represents a hydrogen atom or a hydroxyl group), and a method for producing an anthracycline antibiotic.

Among these anthracycline antibiotics, compounds of formula (I-1)

[Chemical 6] Antibiotics represented by (hereinafter sometimes abbreviated as "S1 substance") and formula (I-3)

[Chemical 7] The antibiotic shown by (hereinafter, may be abbreviated as "S3 substance") is a known substance corresponding to CG15B and CG15A described in Japanese Patent No. 2581931, respectively.

Further, the formula (I-2)

[Chemical 8] The antibiotic indicated by (hereinafter sometimes abbreviated as "S2 substance") is a novel substance not yet published in the literature.

These anthracycline antibiotics are produced by inoculating a nutrient medium with the above transformant and aerobically culturing. The method for culturing the above transformant is based on the method for culturing general microorganisms in principle, but it is usually preferable to carry out under aerobic conditions such as shaking culture by liquid culture and aeration and stirring culture.

The medium that can be used for the culture is
Any medium can be used as long as it contains a nutrient source that can be utilized by a microorganism belonging to actinomycetes such as Streptomyces, and various synthetic media, semi-synthetic media, natural media and the like can be used. As the medium composition, for example, as a carbon source, glucose, maltose, sucrose, starch syrup, molasses, dextrin, starch, glycerol, animal oil, vegetable oil or the like can be used alone or in combination, and as a nitrogen source, Soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, fish meal, urea, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium phosphate and the like can be used alone or in combination. If necessary, an inorganic salt such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, phosphate or metal salt can be added. In addition, organic substances such as vitamins and amino acids that promote the growth of the above strains or the production of the anthracycline antibiotic of the present invention can be added. Further, if necessary, a defoaming agent such as Adecanol (Asahi Denka Kogyo Co., Ltd.) or silicone (Shin-Etsu Chemical Co., Ltd.) can be added. In order to prevent the plasmid from falling off, it is advisable to add an antibiotic such as kanamycin to the medium in an amount of 1 to 10 μg / ml.

The culture conditions may be appropriately selected within a range in which the transformant can grow well and produce the anthracycline antibiotic. For example, the pH of the medium is 5-9.
It is preferably about 6 to 7, usually. The culture temperature is a temperature at which the microorganism grows well, usually 20 to 40 ° C, preferably
It is good to keep at 25-28 ℃. Cultivation time may be about 2 to 8 days, preferably 4 to 5 days. Of course, the various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, etc., and the optimum conditions can be selected and adjusted from the above range accordingly. At that time, the culture solution obtained by stopping the culture when the accumulated amount of the target substance in the culture solution becomes maximum may be used in the following purification step.

The above-mentioned anthracycline antibiotics accumulated in the culture medium thus obtained are filtered,
The cells can be separated from the filtrate or the supernatant by separating the cells by a general solid-liquid separation means such as centrifugation. It can also be recovered from the separated bacterial cells. When recovered from the filtrate or supernatant, first, at pH 7-9, chloroform, toluene, extracted with an organic solvent such as ethyl acetate, further Amberlite XAD (Rohm and Haas Co.), Diaion HP-20 ( It can be treated by column chromatography using a carrier such as polystyrene-based adsorption resin such as Mitsubishi Chemical Co., Ltd.), silica gel, alumina, activated carbon or the like. The method of eluting the target substance from these carriers varies depending on the type and properties of the carrier, but for example, in the case of a polystyrene-based adsorption resin, hydrous alcohol, hydrous acetone or the like can be used as the elution solvent.

Further, gel filtration with Sephadex LH-20 (Pharmacia), Bio-gel P-2 (Bio-Rad), thin layer chromatography with silica gel, alumina, etc., normal phase or reversed phase column Can be used for preparative high performance liquid chromatography (preparative HPLC), etc., and these methods can be separated or purified by using these methods alone or in an appropriate combination and, if necessary, repeatedly used. When recovered from the separated bacterial cells, it is extracted and concentrated using a suitable organic solvent such as acetone, methanol, ethanol or butanol, and then re-extracted with chloroform, ethyl acetate, etc., and isolated and purified in the same manner as above. can do.

The above-mentioned anthracycline antibiotics obtained as described above have the following physicochemical properties. 1.Physicochemical properties of S1 substance (1) Color and shape: Red powder (2) Molecular formula: C ₃₂ H ₃₉ NO ₁₃ (3) Mass spectrum (FAB-MS (m / z)): 646 (M + H ) ⁺ (4) Specific rotation: [α] _D ²⁰ -40 ° (c 0.01, chloroform)

(5) Ultraviolet absorption spectrum: The characteristic absorptions measured in methanol are as follows. UV λ max nm, (ε ^1% _1cm ): 235 (585), 254 (347), 293 (99), 4
95 (207) (6) Infrared absorption spectrum (KBr method): The main absorptions are as follows. ν (cm ⁻¹ ): 1600 (7) ¹ H-NMR spectrum (500 MHz): Chemical shifts and multiplicities of major signals are shown in Table 1. (8) ¹³ C-NMR spectrum (125 MHz): Chemical shifts of major signals are shown in Table 2.

2. Physicochemical properties of S2 substance (1) Color and shape: Red powder (2) Molecular formula: C ₄₀ H ₅₄ N ₂ O ₁₄ (3) Mass spectrum (FAB-MS (m / z)): 787 (M + H) ⁺ (4) Specific rotation: [α] _D ²⁵ + 140 ° (c 0.005, methanol)

(5) Ultraviolet absorption spectrum: The characteristic absorptions measured in methanol are as follows. UV λ max nm, (ε ^1% _1cm ): 235 (436), 254 (259), 292 (86), 4
95 (167) (6) Infrared absorption spectrum (KBr method): The main absorptions are as follows. ν (cm ⁻¹ ): 3450,2936,1601 (7) ¹ H-NMR spectrum (500 MHz): Chemical shifts and multiplicities of major signals are shown in Table 1. (8) ¹³ C-NMR spectrum (125 MHz): Chemical shifts of major signals are shown in Table 2.

3. Physicochemical properties of S3 substance (1) Color and shape: Red powder (2) Molecular formula: C ₃₂ H ₃₉ NO ₁₂ (3) Mass spectrum (FAB-MS (m / z)): 630 ( M + H) ⁺ (4) Specific rotation: [α] _D ²⁰ + 9 ° (c 0.01, chloroform)

(5) Ultraviolet absorption spectrum: The characteristic absorptions measured in methanol are as follows. UV λ max nm, (ε ^1% _1cm ): 234 (651), 254 (382), 293 (123),
495 (236) (6) Infrared absorption spectrum (KBr method): The main absorptions are as follows. ν (cm ⁻¹ ): 1590 (7) ¹ H-NMR spectrum (500 MHz): Chemical shifts and multiplicities of major signals are shown in Table 1. (8) ¹³ C-NMR spectrum (125 MHz): Chemical shifts of major signals are shown in Table 2.

[0039]

[Table 1]

[0040]

[Table 2]

The anthracycline antibiotic of the present invention is
Each of them shows a growth inhibitory effect on cultured leukemia K562 cells and is useful as an autologous cancer drug. (Proliferation inhibitory effect on human leukemia K562 cells) K562 cells were inoculated to RPMI1640 medium (GIBCO BRL; without phenol red) containing 10% fetal calf serum at 2.2 × 10 ⁴ cells / ml,
90 μl was dispensed into a 96-well microtiter plate. A solution of the compound of the present invention was prepared at 10 times the target final concentration.
Add each μl and incubate at 37 ℃ in a carbon dioxide incubator (5% CO _{2 in} air).
The cells were cultured for 75 hours in the atmosphere) to determine the 50% growth inhibitory concentration relative to the control group. In addition, 3 hours before the end of culture,
10 μl of WST-1 (Wako Pure Chemical Industries, Ltd.) was added. Using a microplate reader, the absorbance at 450 nm (control wavelength 650 nm) was measured to count living cells, and the 50% growth inhibitory concentration of each substance for K562 cells was calculated. The results are shown in Table 3.

[0042]

[Table 3]

[0043]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Further, in the following description, the indicated concentration is weight% unless otherwise specified.

Example 1: Isolation and identification of rhoG gene (1) Streptomyces v
Preparation of genomic DNA of iolaceus) A262, SC-7 YGS medium (soluble starch 0.5%, yeast extract 0.3%, glucose 0.5%, pH 7.2) 4 ml Streptomyces violaceus (Streptomyces violaceus) A262, SC-7 Inoculate,
It was cultured at 28 ° C for 3 days. The obtained culture solution was centrifuged at 3000 rpm for 10 minutes to collect bacterial cells. Blood & Cell Cul from the fungus body
Genomic DNA was prepared using a ture kit (QIAGEN).

(2) Cloning of partial sequence of DNA encoding rhodosamine transferase J. Bacteriol., 177, 6688-6692 (1995) and Proc. Natl. Ac
Based on the description of ad.Sci.USA, 95,12111-12116 (1998), etc.
Amino acid sequences of glycosyltransferases derived from microorganisms that produce anthracycline antibiotics or macrolide antibiotics are highly conserved amino acid sequences (Val Pro Leu Ala Trp Ala Leu and Ala Val Val
From His His Gly) to the following primers (P1 and P1
2) was designed and created (see SEQ ID NOS: 2 and 3 in the sequence listing). P1 5'-TGGTGCCGCTGGCCTGGGCGCTGC-3 'P2 5'-CCGCCGTGGTGCACGACCGCCG-3'

Next, these two primers (P1 and P1
2) and Streptomyces violaceus obtained in the previous section (1)
PCR was performed using the genomic DNA of (Streptomyces violaceus) A262, SC-7 as a template. The PCR reaction is
Takara LA Taq with GC Buffer (Takara Shuzo) and PCR amplification device Program Temp Control System PC-700 (ASTEC)
Was performed under the following conditions.

(Composition) LA Taq (5units / ml) 0.5 μl 2 x GC Buffer I 25 μl A262, SC-7 Genomic DNA 50ng P1 primer 10pmol P2 primer 10pmol Sterile distilled water up to 50 μl

(Time & cycle) 94 ℃ 5 minutes 1 cycle 98 ℃ 20 seconds & 72 ℃ 1 minute 35 cycle 72 ℃ 5 minutes 1 cycle

As a result, a DNA fragment having a size of about 900 bp (hereinafter referred to as DNA fragment-A) was amplified. Then, the reaction solution containing the DNA fragment-A amplified by the PCR reaction was subjected to agarose gel electrophoresis for fractionation. The DNA fragment-A having a size of about 900 bp was cut out from an agarose gel and recovered by SUPREC 01 (Takara Shuzo).

Next, in order to obtain a sufficient amount of DNA fragment-A for analyzing the nucleotide sequence of the obtained DNA fragment-A, the plasmid vector pT7Blue T (Novagen) was used for DNA Ligation kit ve.
The DNA fragment-A was ligated using r.2 (Takara Shuzo) and E. coli DH
5a was transformed. Then ampicillin (50 μg / ml),
X-gal (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside)
40 μg / ml, IPTG (Isopropyl-β-D-thiogalactopyranosid
e) LB agar containing 100 μM (1.0% bactotryptone, 0.
Transformed E. coli was selected using 5% yeast extract, 1% NaCl, pH 7.0, 1.5% agar). The colonies of the transformed Escherichia coli thus separated were cultured in an LB liquid medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) containing 50 μg / ml of ampicillin. Plasmid DNA was extracted from each of the grown transformed Escherichia coli cells, the resulting plasmid DNA was treated with restriction enzymes EcoRI and HindIII, and the DNA size was measured using agarose electrophoresis. About 900 bp with about 2.9 kbp from T
A plasmid from which a DNA fragment showing the size of
The plasmid pRG9) was selected.

(3) Analysis of the base sequence of the cloned DNA fragment-A The base sequence of the DNA fragment-A obtained in (2) above was analyzed using a DNA base sequence analyzer PE Biosystems 373S (Applied Biosystems). It was analyzed by the dye terminator cycle sequence method. As a result of the nucleotide sequence analysis, the DNA fragment-A amplified by the PCR reaction was measured by electrophoresis to be about 900 bp, but the nucleotide sequence analysis revealed that it was exactly 928 bp (SEQ ID NO: 1). See base 987 to base 1868). The base number is that of the corresponding sequence without the primer sequence. Since DNA sequences corresponding to the two types of primers used in the PCR reaction were found at both ends of the cloned 928 bp DNA sequence, in the PCR reaction,
It was revealed that DNA fragment-A was specifically amplified by these two kinds of primers (P1 and P2).

(4) Analysis of the peripheral region of DNA fragment-A by inverse PCR method As described above, Streptomyces violaceus (Str
eptomyces violaceus) A262, the partial sequence of the DNA encoding the rhodosamine transferase from SC-7 was determined, so the inverse PCR method (Cell engineering 14: p.591-593, 1995) The nucleotide sequence of the peripheral region spread in the downstream region was amplified and cloned, and the sequence was analyzed. That is, Streptomyces violaceus
(Streptomyces violaceus) A262, SC-7 genomic DNA to L buf
It was fully digested with the restriction enzyme SacI in fer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM dithiothreitol, 100 mM NaCl). The obtained restriction enzyme-cut DNA fragment is used as a DNA Ligation Kit
It was self-cyclized using ver.2 (Takara Shuzo).

On the other hand, the following two sets of primers (A1 and A2) and (B1 and B2) were designed and prepared from the nucleotide sequence of DNA fragment-A (see SEQ ID NOS: 4 to 7 in the sequence listing). (5 'side) A1 5'-TGATGCTCTCGACCGCGGCCGGCTGGCCGG-3' A2 5'-CTGCGGGCGTCGAACGCGATGCTGACGGCG-3 '(3' side) B1 5'-AGAGCACCAGGTCCGGCTGCCAGAACCGGG-3 'B2 5'-ACAACACGCGGGTGGTCGACCACGTGGCCC

Next, these two sets of primers ((A1 and A2) and (B1 and B2)) and the self-circularized Streptomyces violaceus A262, SC
PCR reactions were performed separately using -7 genomic DNA as a template (see Figure 1). Takara LA Taq wit for each PCR reaction
h GC Buffer (Takara Shuzo) and PCR amplification device Program TempCo
ntrol System PC-700 (manufactured by ASTEC) was used under the following conditions.

(Composition) LA Taq (5 units / ml) 0.5 μl 2 x GC Buffer I 25 μl Circularization A262, SC-7 Genomic DNA 50ng A1 primer (or B1 primer) 10pmol A2 primer (or B2 primer) 10pmol Sterile distilled water up to 50 μl

(Time & cycle) 94 ℃ 5 minutes 1 cycle 98 ℃ 20 seconds & 72 ℃ 3 minutes 35 cycle 72 ℃ 5 minutes 1 cycle

As a result, a DNA fragment (D
NA fragment-B) and a DNA fragment of about 2.4 kbp (DNA fragment-C)
Was amplified (see FIG. 2). These are the DNA encoding the rhodosamine transferase and the DNA containing its upstream and downstream regions.
It is likely that the DNA has a sequence.

This PCR amplification reaction solution was subjected to agarose gel electrophoresis for fractionation. DNA fragments of about 1.2 kbp and about 2.4 kbp were excised from agarose gel and recovered by SUPREC 01 (Takara Shuzo). Next, for the obtained DNA fragment-B and DNA fragment-C, in order to obtain a sufficient amount of each DNA fragment for analyzing the nucleotide sequence, plasmid vector pT7Blue T (Novagen), DN was used in the same manner as in (2) above.
A Ligation kit ver.2 (Takara Shuzo), E. coli DH5a and plasmid purification kit QIAfilter Plasmid Midi Kit (QIA
GEN) was used to obtain a fixed amount of each DNA fragment.

(5) DNA fragment-B (size of about 1.2 kbp) and
Analysis of nucleotide sequence of DNA fragment-C (size of about 2.4 kbp) The nucleotide sequences of DNA fragment-B and DNA fragment-C obtained in the previous section (4) were analyzed using PE Biosystems 373S (Applied
Biosystems) and analyzed by the dye terminator cycle sequence method. In this way, the base sequence was analyzed, and from the DNA fragment-B and DNA fragment-C sequences, information on the 4133 bp base sequence shown in SEQ ID NO: 1 in the sequence listing described below was obtained. A frame plot analysis of this nucleotide sequence revealed two complete open reading frames (ORFs) and two incomplete ORFs lacking the 5'side or 3'side.
Was found to exist.

Among these ORFs, the ORF encoding a protein consisting of 426 amino acids located from base 914 to base 2194 (hereinafter referred to as rhoG) was found to be in the anthracycline antibiotic biosynthesis system as a result of homology search. High homology (homology 50 to 60%) was observed with a family of glycosyltransferases, for example, a gene encoding a glycosyltransferase derived from a nogaramycin- or aclarubicin-producing bacterium. Among them, it showed the highest homology (60% homology) with the deduced amino acid sequence of the gene (dnrS) encoding a daunosamine transferase derived from the daunomycin-producing bacterium Streptomyces peucetius ATCC12520. This suggests that rhoG is likely to encode an enzyme capable of transferring rhodosamine to ε-rhodomycinone.

In addition, 327 located between base 2217 and base 3200
ORF (hereinafter referred to as ORF-2), which encodes a protein consisting of 4 amino acids, is dTDP-glucose found in sugar chain biosynthesis of many antibiotics including the anthracycline system.
It showed a high homology of about 65% with the se-4,6-dehydratase family. An incomplete ORF lacking the 5'side (hereinafter referred to as ORF-1) is located from base 1 to base 879 and encodes 292 amino acids, and its deduced amino acid sequence is a sugar chain of an anthracycline antibiotic. Streptomyces galileaeus (S
Treptomyces galilaeus) AknT and Streptomyces peucetius DnrQ had 40% and 43% homology, respectively.

Also located at bases 3276 to 4133 is 285
Another incomplete ORF (hereinafter referred to as ORF-3), which encodes 1 amino acid and lacks the 3'side, is responsible for the autoresistance mechanism of anthracycline antibiotic-producing bacteria ABC (AT
P biding casstte) transporter family and 50% ~ 6
There was 0% homology.

(6) Isolation of full-length rhoG gene The same operation as in (1) was performed to prepare genomic DNA of A262, SC-7 strain. Using this genomic DNA as a template, PCR reaction was carried out using the following primers (R1 and R2) containing the start and stop codons of rhoG (see SEQ ID NOs: 8 and 9 in the sequence listing). The PCR reaction is based on Takara LA Taq with GC Buffer
(Takara Shuzo) and PCR amplification device Program Temp Control Syst
The emPC-700 (ASTEC) was used under the following conditions. R1 5'-CCCAAGCTTGTGCGTGTACTGCTGACCGC-3 'R2 5'-CCGGAATTCTCAGCGAGCCGCGACCGGCG-3'

(Composition) LA Taq (5 units / ml) 0.5 μl 2 x GC Buffer I 25 μl A262, SC-7 strain Genomic DNA 50ng R1 primer 10pmol R2 primer 10pmol Sterile distilled water up to 50 μl

(Time & cycle) 94 ℃ 5 minutes 1 cycle 98 ℃ 20 seconds & 72 ℃ 1 minute 35 cycle 72 ℃ 5 minutes 1 cycle

As a result, a DNA fragment having a size of about 1.2 kbp was amplified. Next, the DNA fragment-A amplified by the PCR reaction was treated with restriction enzymes EcoRI and HindIII, and the reaction solution was subjected to agarose gel electrophoresis for fractionation. This DNA fragment of about 1.2 kbp was cut out from the agarose gel and
Recovered by REC 01 (Takara Shuzo).

In order to obtain a fixed amount of the DNA fragment containing the full-length rhoG gene thus obtained, this DNA fragment was prepared using plasmid vector pUC19 (Takara Shuzo) with DNA Ligation kit ver.2 (Takara Shuzo). Were ligated and E. coli DH5a was transformed. Thereafter, ampicillin 50 μg / ml, X-gal
LB agar medium containing 40 μg / ml, IPTG 100 μM (1.0% bactotryptone, 0.5% yeast extract, 1% NaCl, pH 7.0, 1.5%
The transformed E. coli was selected using (agar). Transformed E. coli colonies thus isolated were treated with ampicillin 50
LB liquid medium containing μg / ml (1% bactotryptone, 0.5%
It was cultured in yeast extract, 1% NaCl, pH 7.0). Plasmid DNA was extracted from each of the grown transformed Escherichia coli cells, and the plasmid DNA obtained was extracted with restriction enzymes EcoRI and Hind.
Select a plasmid (hereinafter referred to as plasmid pRG02) that is treated with III and obtained a fragment showing a size of about 1.2 kbp together with about 2.7 kbp derived from plasmid pUC19 when the size of DNA is measured using agarose electrophoresis. did.

(7) Preparation of rhoG Gene Expression Transformant and Expression Vector Next, the actinomycete plasmid pIJ6021 (Gene 166,133-137 (199)
5)) was treated with restriction enzymes EcoRI and HindIII, and the reaction solution was subjected to 0.7% agarose gel electrophoresis for fractionation.
This DNA fragment having a size of about 7.8 kbp was cut out from agarose and recovered by SUPRED 01 (Takara Shuzo). A 1.2 kbp DNA fragment was excised and recovered from the plasmid pRG02 by the same treatment with the same restriction enzymes. This 7.8kb
Mix the p and 1.2 kbp fragments and use the DNA Ligation Kit ver.
Ligation reaction was performed using 2 (Takara Shuzo).

200 μl of Streptomyces lividans TK24 protoplast suspension and T buffer (33 mM Tris-H) were added to the above ligation reaction product.
Cl, pH 7.9, 10 mM magnesium acetate, 0.5 mM dithiothreitol, 66 mM potassium acetate) (400 μl, twice the volume of the protoplast suspension) was added and gently mixed. This was mixed with R2YE top agar kept warm at 50 ° C and mixed with R2YE medium (Genetic Ma
nipulation of Streptomyces: A Laboratory Manual. Jo
hn Innes Foundation, Norwich, 1985: Medium components were shown below). After culturing at 30 ℃ for 16-24 hours, keep it at 50 ℃ and add kanamycin (hereinafter abbreviated as "Km").
Layers were overlaid with 2 ml of overlaying agar medium added to 1.5 mg / ml and cultured at 30 ° C for 4 to 5 days. Plasmid DNA was extracted and prepared from the grown kanamycin resistant colonies by a small amount preparation. The obtained plasmid DNA was digested with the restriction enzyme Eco.
Approximately 7. from the plasmid pIJ6021 treated with RI and HindIII and measured for DNA size using agarose electrophoresis.
An expression vector pBRG12 containing a fragment showing a size of about 1.2 kbp together with 8 kbp was selected.

R2YE medium: sucrose 10.3%, potassium sulfate 0.025%, glucose 1%, casamino acid 0.01%, trace tra
ce element solution 0.2% (v / v), yeast extract 0.5%, magnesium chloride hexahydrate 0.12%, TES 25mM, agar 2.2% and 0.5% dibasic potassium phosphate 1% (v / v), 5M chloride Calcium dihydrate 0.4% (v / v), 20% Asparagine 1.5% (v / v), 1
NNaOH (0.7% (v / v) (these were sterilized separately) R2YE overlay medium (R2YE top agar): The agar concentration of the above R2YE medium was changed to 0.7%

Example 2: Preparation of rhoG gene-disrupted strain (1) Preparation of vector for rhoG gene disruption Disruption of glycosyltransferase gene (rhoG) was carried out by SacI in rhoG gene.
Utilizing a frameshift mutation caused by blunt-end (4 base deletion) of the site, and introducing the mutation on the chromosome, double homologous recombination (doubl
e crossover) induction.

That is, a plasmid pRG9 having an internal region of about 900 bp lacking start and stop codons was treated with a restriction enzyme SacI, and the resulting SacI fragment of about 3.6 kbp was blunt-ended to obtain DNA Ligation kit ver. 2 (Takara Shuzo). Was used for self-cyclization. Escherichia coli DH5a was transformed with the reaction product, and ampicillin 50 μg / ml, X-gal 40 μg / ml, IPTG 100
Transformed E. coli was selected using LB agar containing μM. The thus transformed colonies of transformed E. coli were cultured in LB liquid medium containing 50 μg / ml of ampicillin,
Plasmid DNA was extracted from the grown bacterial cells. The plasmid DNA was treated with the restriction enzyme SacI, and a plasmid that was not cleaved by 0.7% agarose electrophoresis, that is, a plasmid having a blunt-ended SacI site (hereinafter referred to as "plasmid pBRG9") was selected.

Then, the actinomycete plasmid pIJ486 (Mol. Gen. G
enet.203,468-478 (1986)) with restriction enzymes EcoRI and HindII.
After treatment with I, the reaction solution was subjected to 0.7% agarose electrophoresis for fractionation. The DNA fragment having a size of about 6.2 kbp was cut out from an agarose gel and recovered by SUPREC 01 (Takara Shuzo). A DNA fragment of about 0.9 kbp was cut out from the plasmid pBRG9 by the same treatment with the same restriction enzymes and recovered. Both DNA fragments are mixed, and DNA ligation is performed.
Ligation reaction was performed using Kit ver.2 (Takara Shuzo).

Using the resulting reaction product, 200 μl of Streptomyces lividans TK24 protoplast suspension and 400 μl of T buffer (2 times the volume of protoplast suspension) were added and mixed gently.
This was mixed with R2YE topagar which was kept warm at 50 ° C and overlaid on R2YE medium. After culturing at 30 ° C for 16 to 24 hours, incubate at 50 ° C and add thiostrepton (hereinafter sometimes abbreviated as "Ts") to 50%.
Overlay with 2 ml of overlay medium added to give μg / ml, 30 ℃
The cells were cultured for 4 to 5 days. Plasmid DNA was prepared from the grown thiostrepton resistant colonies. The resulting plasmid DNA was treated with restriction enzymes EcoRI and HindIII, and a fragment containing a fragment showing a size of 0.9 kbp together with 6.2 kbp derived from the plasmid pIJ486 when the size of the DNA was measured using agarose electrophoresis (hereinafter, , "Gene disruption vector pBRG19") was selected (see FIG. 3).

(2) Selection of gene-disrupted strain Streptomyces viol
aceus) A262, SC-7 was transformed with the gene disruption vector pBRG19 to obtain the SC-7 / pBRG19 strain. SC-7 / pBRG19 strain was protoplasted, regenerated on R2YE medium, and the purified colonies were plated on YGS agar medium (yeast extract 0.3%, soluble starch 0.5%, glucose 0.5%, agar 1.5%, pH 7.2). Transfer 28
It was cultured at ℃ for 5 days. Each colony having a radius of about 5 mm was transferred to a test tube, 0.2 ml of acetone was added and mixed vigorously, and 0.2 ml each of chloroform and water were added and mixed in the same manner.

After centrifugation at 3000 rpm for 10 minutes, the chloroform layer was separated, concentrated to dryness, dissolved in a small amount of chloroform and spotted on silica gel 70F254 plate (Wako Pure Chemical Industries, Ltd.), and chloroform: methanol (15: It was developed with the solvent system of 1). At this time, a strain that produces ε-rhodomycinone with an Rf value of 0.6 was transformed into the vector pBRG19 with A2 by homologous recombination.
62, Single homologous recombination on SC-7 chromosome
(single crossover) strain was selected. By such an operation, from 1000 purified colonies, 5 strains of once homologous recombination which were ε-rhodomycinone-producing and Ts-resistant were obtained.

In order to release the pIJ486 portion containing the Ts resistance marker from the single-homologous recombinant strain, each of these single-homologous recombinant strains was treated with YGS agar medium (yeast extract 0.3%, soluble starch 0.5%) containing no thiostrepton. %, Glucose 0.5%, agar 1.5%, pH 7.2). The sporulated strain was protoplasted, regenerated, and purified colonies were transferred to YGS agar medium and cultured at 28 ° C for 3 days. Furthermore, each grown colony is treated with thiostrepton.
YGS agar medium containing 20 μg / ml was inoculated and cultured at 28 ° C. for 3 days to examine the presence or absence of growth, and a strain that did not grow, that is, a strain exhibiting Ts sensitivity was selected as a double crossover strain. By this procedure, about 1% of the homologous recombinant strains was obtained in the 7th sporulation.
A Ts-susceptible strain (double homologous recombination strain) could be obtained with the probability of.

(3) Analysis of products of gene-disrupted strains Two strains randomly selected from the twice homologous recombinant strains obtained in (2) above were designated as M7-75 strain and M7-154 strain,
Product analysis and detection for mutations in the rhoG gene were performed. YGS medium (yeast extract 0.3%, soluble starch 0.5%,
A262, SC-7 strain, M7-75 strain and M7-154 strain were respectively inoculated into 3 test tubes sterilized by dispensing 4 ml of glucose 0.5%, pH 7.2) and cultured at 28 ° C for 3 days. 3000 rpm of the obtained culture solution,
Collect the cells by centrifugation for 10 minutes, add 0.2 ml of acetone to this, mix vigorously, and add 0.2 ml each of chloroform and water.
Each was added and mixed in the same manner. After centrifugation at 3000 rpm for 10 minutes, the chloroform layer was separated and concentrated to dryness. It was dissolved in a small amount of chloroform, spotted on a silica gel 70F254 plate (Wako Pure Chemical Industries, Ltd.), and developed with a solvent system of chloroform: methanol: ammonia water (8: 1: 0.5). The results are shown in Fig. 4. The A262, SC-7 strain produces the glycosides rhodomycins (Rf value: 0.2 to 0.4), whereas M7-75
Strain and M7-154 strain are both ε-rhodomycinone (Rf value: 0.8)
Accumulated.

(4) Analysis of mutation site in gene-disrupted strain The same operation as in Example 1 (1) was performed to carry out strains M7-75 and M7-154.
The strain's genomic DNA was prepared. As a control, genomic DNA of A262, SC-7 strain was also prepared. Using these genomic DNAs as templates, PCR reactions were performed using the following primers (R1 and R2) containing the start and stop codons of rhoG (see SEQ ID NOS: 8 and 9 in the sequence listing). PCR reaction is Takara LA Ta
q with GC Buffer (Takara Shuzo) and PCR amplification device Program T
It was carried out under the following conditions using emp Control System PC-700 (ASTEC). R1 5'-CCCAAGCTTGTGCGTGTACTGCTGACCGC-3 'R2 5'-CCGGAATTCTCAGCGAGCCGCGACCGGCG-3'

(Composition) LA Taq (5 units / ml) 0.5 μl 2 x GC Buffer I 25 μl M7-75 strain or M7-154 strain Genomic DNA 500ng R1 primer 110pmol R2 primer 210pmol Sterile distilled water up to 50 μl

(Time & cycle) 94 ℃ 5 minutes 1 cycle 98 ℃ 20 seconds & 72 ℃ 1 minute 35 cycle 72 ℃ 5 minutes 1 cycle

Each amplification product was treated with the restriction enzyme SacI to obtain 0.7
Electrophoresed on a% agarose gel. The migration pattern is shown in FIG. In the A262 and SC-7 strains, bands of about 0.3 kbp and about 0.9 kbp appeared due to the presence of the SacI cleavage site.
In the -75 strain and the M7-154 strain, the SacI cleavage site in the A262 and SC-7 strains was blunt-ended and disappeared, so only a band of about 1.2 kbp was confirmed. From this, the M7-75 strain and M7-
In strain 154, it is clear that the function of the rhoG gene was disrupted by frameshifting due to the blunt end of the SacI cleavage site.

Example 3: Isolation of dnrS gene and construction of expression vector (1) Isolation of dnrS gene The same operation as in Example 1 (1) was performed, and Streptomyces peucetius ATCC12520 was performed.
The strain's genomic DNA was prepared. Then J. Bacteriol. 177,6
Referring to the description in 688-6692 (1995), HindII is added to the start codon side.
The following primers (DS1 and DS2) were designed and prepared so as to have an EcoRI site on the I site and stop codon side (see SEQ ID NOS: 10 and 11 in the sequence listing). DS1 5'-CCCAAGCTTATGAAGGTGCTCGTGACGGC-3 'DS2 5'-CCGGAATTCCTAGTGCCGGACGCCCTGCC-3'

These two kinds of primers (DS1 and DS2) and Streptomyces pucetius (Strepto
PCR was performed under the same conditions as in Example 1 (2) using the genomic DNA of myces peucetius) ATCC12520 strain as a template. The obtained 1.3 kbp amplification product was treated with restriction enzymes EcoRI and HindIII and fractionated by agarose electrophoresis to obtain a dnrS gene encoding daunosamine transferase. Then, in the 2.7 kbp of plasmid pUC19 (GENE 33,103-119 (1985)) which had been previously treated with restriction enzymes EcoRI and HindIII.
The DNA fragment containing the dnrS gene was ligated using DNA Ligation kit ver.2 (Takara Shuzo), and Escherichia coli DH5a was transformed. By the same procedure as in Example 1 (2), the transformant was grown, the plasmid was extracted, the restriction enzyme treatment was performed, and the plasmid was selected, and a certain amount of the plasmid containing the 1.3 kbp dnrS gene together with 2.7 kbp derived from pUC19 (hereinafter "Plasmid pDS1
3 ”).

(2) Incorporation of dnrS gene into actinomycete expression plasmid Actinomycete plasmid pIJ6021 (Gene 166, 133-137 (1995)) was treated with restriction enzymes EcoRI and HindIII, and the reaction solution was treated with 0.
Fractionation was performed by 7% agarose gel electrophoresis. This about
A DNA fragment with a size of 7.8 kbp was cut out from an agarose gel and recovered by SUPREC 01 (Takara Shuzo). Previous paragraph
A 1.3 kbp DNA fragment containing the dnrS gene was excised from the plasmid PD13 prepared in (1) by the same restriction enzyme treatment and technique, and was recovered. This about 7.8kbp and 1.3kbp DN
The A fragments were mixed and subjected to ligation reaction using DNA Ligation Kit ver.2 (Takara Shuzo).

200 μl of Streptomyces lividans TK24 protoplast suspension and 400 μl of T buffer (2 times the volume of protoplast suspension) were added to this reaction product and mixed gently. This was mixed with R2YE top agar kept warm at 50 ° C. and overlaid on the R2YE medium. After culturing at 30 ℃ for 16-24 hours, keep it at 50 ℃ and add kanamycin to the concentration of 1.5 mg / ml (Nutrient br
oth 0.8%, agar 0.4%, pH 7.2) Overlay with 2 ml, 4 ~ 30 ℃
Cultured for 5 days. Plasmid DNA was extracted and prepared from the grown Km resistant colonies by a small amount preparation. The obtained plasmid DNA was treated with restriction enzymes EcoRI and HindIII, the size of the DNA was measured using agarose electrophoresis, and a DNA fragment having a size of 1.3 kbp together with 7.8 kbp from pIJ6021 was prepared. "Plasmid pDS23") was selected.

Example 4: Introduction of dnrS gene expression vector into rhoG gene-disrupted strain The same procedure as in Example 3 (2) was performed to prepare rho prepared in Example 2.
The G gene-disrupted strain M7-75 strain was transformed with the plasmid pDS23, which is a dnrS gene expression vector. Plasmid DNA was extracted from the grown Km resistant strain, and it was confirmed that the plasmid pDS23 was retained. Thus the plasmid pDS23
Streptomyces violaceus (S
Treptomyces violaceus) M7-75 / pDS23 strain was obtained.

Example 5: Culture of Streptomyces violaceus M7-75 / pDS23 strain (1) Seed culture Seed culture medium (soluble starch 0.5%, yeast extract 0.1%, soybean flour 1.0%, glucose 0.5%, maltose 2.0%, sodium chloride 0.1%, antifoaming agent (10% Adecanol (Asahi Denka Co.)) 0.1% (v / v), magnesium sulfate heptahydrate 0.1%,
Di-potassium phosphate hydrate 0.1%, pH 7.0, all (w / v) except antifoaming agent) (50/500 ml Sakaguchi flask containing 50 ml of M7-75 / pDS23 strain from slope culture. Shirokane ear inoculation, 30
Seed culture was performed by shaking culture at ℃ for 3 days. Note that kanamycin was added at 5 μg / ml at the start of seed culture.

(2) Production culture The production culture is a production medium (soluble starch 4.0%, yeast extract 0.2%, soybean flour 2.5%, salt 0.25%, calcium carbonate 0.
4%, defoamer (10% ADECANOL (Asahi Denka Co.)) 0.1% (v /
v), zinc sulfate heptahydrate 0.0002%, ferric sulfate heptahydrate
0.0001%, copper sulfate heptahydrate 0.0007%, manganese sulfate-7
Hydrate 0.0003%, pH 7.0, all (w / v) except defoamer. Calcium carbonate is added after adjusting the pH. 60 ml of a 500 ml Sakaguchi flask in which 50 ml of the production medium was dispensed and sterilized was inoculated with 1 ml of the seed mother prepared in (1) above, and cultured at 28 ° C for 6 days with shaking. At the start of seed culture, kanamycin was added at 5 μg / m
l, thiostrepton was added on the second day of culture so as to be 5 μg / ml. Thus, 3000 ml of the culture solution was prepared.

Example 6: Extraction and purification of anthracycline antibiotics S1 to S3 Streptomyces violaceus obtained in Example 5 (2)
(Streptomyces violaceus) M7-75 / pDS23 strain culture medium 3000
The cells were centrifuged at 3000 rpm for 10 minutes at room temperature to separate into bacterial cells and culture supernatant. Add 1000 ml of chloroform to the culture supernatant,
After vigorous stirring with a separating funnel, a chloroform layer (derived from the culture supernatant) was obtained by centrifugation. On the other hand, 1000 ml of acetone was added to the cell division, and the mixture was stirred with a magnetic stirrer for 5 minutes and then centrifuged. The acetone layer of the supernatant was concentrated under reduced pressure by a rotary evaporator to about 1/2 volume,
400 ml of water and 300 ml of chloroform were added, and the mixture was vigorously stirred with a separating funnel and then centrifuged to obtain a chloroform layer (derived from bacterial cell division). The culture supernatant and each chloroform layer derived from the cell division were mixed and concentrated under reduced pressure to a small amount, and then excess n-hexane was added to precipitate the extract. The precipitate was collected by centrifugation and then vacuum dried with a desiccator to obtain about 400 mg of a crude extract.

The crude extract was purified by silica gel column chromatography. About 400 mg of the crude extract was dissolved in a small amount of chloroform, and a silica gel column (φ containing Wakogel C-200 (Wako Pure Chemical Industries) was previously packed with chloroform.
29 × 100 mm). After this, chloroform: methanol: water (100: 10: 0.1, 80: 10: 0.1, 60: 10: 0.1)
Elution was carried out stepwise with 300 ml each. The obtained fraction was analyzed using a silica gel 70F254 plate (Wako Pure Chemical Industries, Ltd.) in a solvent system of methanol: chloroform: ammonia water (8: 1: 0.5), and the products S1, S2 and S3 were respectively analyzed. The contained fractions were collected and concentrated to dryness to obtain each partially purified product.

The final purification of S1 to S3 was carried out by the scraping method by TLC. Each partially purified product that had been concentrated to dryness was dissolved in a small amount of chloroform, and applied to the silica gel 70F254 plate (Wako Pure Chemical Industries, Ltd.) in a band shape at the lower end 20 mm,
Air dry, chloroform: methanol: ammonia water
It was developed in a solvent system of (8: 1: 0.5). Scrap the dye band corresponding to each product, and use methanol: ammonia water (10
It was eluted at 0: 1) and concentrated to dryness. Then, add these to chloroform: methanol: water: acetic acid: ammonia water (120: 5
It was developed in a solvent system of 0: 5: 1: 1). Each dye band was scraped off from the silica gel plate, eluted with methanol: ammonia water (100: 1) and concentrated to dryness. Distilled water was added to each concentrate, chloroform was further added, and the mixture was extracted with stirring. The chloroform layer was separated and concentrated by an evaporator, and an excess amount of n-hexane was added to this to cause precipitation, which was collected and dried. Finally purified pure S1, S2, S3 4.7 mg, 1
Obtained 5.0 mg and 2.5 mg.

[0093]

[Sequence list]                         SEQUENCE LISTING <110> Mercian Corporation <120> rhoG gene and its use <130> H13-0892 <160> 11 <210> 1 <211> 4133 <212> DNA <213> Streptomyces violaceus <220> <221> CDS <222> (1) .. (876) <220> <221> CDS <222> (914) .. (2191) <220> <221> CDS <222> (2217) .. (3197) <220> <221> CDS <222> (3276) .. (4133) <400> 1 gag ctc gcc gag gtc tcc gcg ccg ttg ctg tcg gcc gag gcg ctc gcg 48 Glu Leu Ala Glu Val Ser Ala Pro Leu Leu Ser Ala Glu Ala Leu Ala   1 5 10 15 gcg cgg gcg cct cgg atc gag gcg gcc tac cgc gag acg ctc gcg gcg 96 Ala Arg Ala Pro Arg Ile Glu Ala Ala Tyr Arg Glu Thr Leu Ala Ala              20 25 30 acg ggc gaa cgg ttc gac ctg tac cgg gac ttc gcc gtc ccc gcc gcc 144 Thr Gly Glu Arg Phe Asp Leu Tyr Arg Asp Phe Ala Val Pro Ala Ala          35 40 45 ctg cgg gtg gtg gcc gat gtg ctc ggc ctg ccc gcc gag acc cgc gag 192 Leu Arg Val Val Ala Asp Val Leu Gly Leu Pro Ala Glu Thr Arg Glu      50 55 60 cgc ctg ccc gaa ctg cac gcg ggc ctc gcc acc gcc ctg gac gcc ggc 240 Arg Leu Pro Glu Leu His Ala Gly Leu Ala Thr Ala Leu Asp Ala Gly  65 70 75 80 cac tgc ccg cag cag ctg ggg acc gcc cgg gcg ctg ctc acg gcc gtg 288 His Cys Pro Gln Gln Leu Gly Thr Ala Arg Ala Leu Leu Thr Ala Val                  85 90 95 tcg ggg ctg tcc gcc gag ctg acc ggc ctg gtg gac acc ggc cgc gcc 336 Ser Gly Leu Ser Ala Glu Leu Thr Gly Leu Val Asp Thr Gly Arg Ala             100 105 110 acc ggc ctc gcc ggt ctg tcg ccc gag gac gcg gtc gcg atc ggc gcg 384 Thr Gly Leu Ala Gly Leu Ser Pro Glu Asp Ala Val Ala Ile Gly Ala         115 120 125 ctc gcc gtc gtg gtg ggc gtc gag gtc acg gcg acc gcc gtg tgc aac 432 Leu Ala Val Val Val Gly Val Glu Val Thr Ala Thr Ala Val Cys Asn     130 135 140 gcc gtc gag gtc ctg ctg gcg cac ccg gag cag tgg cag cgg atc ggg 480 Ala Val Glu Val Leu Leu Ala His Pro Glu Gln Trp Gln Arg Ile Gly 145 150 155 160 cag gac ccg gcg gcg gcc ccg gcc gcg gtc cgg gag gcc ctg cgg ctg 528 Gln Asp Pro Ala Ala Ala Pro Ala Ala Val Arg Glu Ala Leu Arg Leu                 165 170 175 gct ccg ccg ctg cgg ttc gag agc cgt atc gcc cgc gag cag gtc gag 576 Ala Pro Pro Leu Arg Phe Glu Ser Arg Ile Ala Arg Glu Gln Val Glu             180 185 190 ttc gac ggc aag acg atc gag gag ggc cgc cgc gtg gtg gtg ctc gtc 624 Phe Asp Gly Lys Thr Ile Glu Glu Gly Arg Arg Val Val Val Leu Val         195 200 205 gac gcg gcc aac cgg gac ccg gag gcc ttc gcc gac ccg gac gcc ttc 672 Asp Ala Ala Asn Arg Asp Pro Glu Ala Phe Ala Asp Pro Asp Ala Phe     210 215 220 acg ccc ggc cgc ggc gac ggc gac cgg tcg ctc acc ctc ctc ggc ggc 720 Thr Pro Gly Arg Gly Asp Gly Asp Arg Ser Leu Thr Leu Leu Gly Gly 225 230 235 240 ccc gcc gcc ccg gtc acc gac gcg ctg gtc cgc gtc cag acc gag gcc 768 Pro Ala Ala Pro Val Thr Asp Ala Leu Val Arg Val Gln Thr Glu Ala                 245 250 255 gcg gtc cgc gcc ctg gcc gca ctc cgg ccc gcc ctg cgc acg aac gcc 816 Ala Val Arg Ala Leu Ala Ala Leu Arg Pro Ala Leu Arg Thr Asn Ala             260 265 270 gag gta ctg cac cgc atg cgc tcg gcg gtg ctg cac ggc gtc ctg cgc 864 Glu Val Leu His Arg Met Arg Ser Ala Val Leu His Gly Val Leu Arg         275 280 285 ttc ccc gtc gca tgaccggacc ggcccacacc aggggaggag caccgcg gtg cgt 919 Phe Pro Val Ala Met Arg     290 gta ctg ctg acc gcc ttc gca cag gac gcc cat ctc aac ggc gtc gtc 967 Val Leu Leu Thr Ala Phe Ala Gln Asp Ala His Leu Asn Gly Val Val 295 300 305 310 ccg ctc gcc tgg gcg ctg cgg acc gcc ggg cac gag gtc cgg gtg gcc 1015 Pro Leu Ala Trp Ala Leu Arg Thr Ala Gly His Glu Val Arg Val Ala                  315 320 325 ggc cag ccg gcc gcg gtc gag agc atc acc cgg gcc ggt ctg acg gcc 1063 Gly Gln Pro Ala Ala Val Glu Ser Ile Thr Arg Ala Gly Leu Thr Ala             330 335 340 gtt ccg gtg ggc gac aac cac cgg atg gac gcc gtc atc ccc acc gtc 1111 Val Pro Val Gly Asp Asn His Arg Met Asp Ala Val Ile Pro Thr Val         345 350 355 ggt ccc ggg atg ctc atg cac cac ctg gac cgc gac tat ctg gag aac 1159 Gly Pro Gly Met Leu Met His His Leu Asp Arg Asp Tyr Leu Glu Asn     360 365 370 cgt ccc gag cgg ctg agc ctc gac ttc ctg cgg gcg tcg aac gcg atg 1207 Arg Pro Glu Arg Leu Ser Leu Asp Phe Leu Arg Ala Ser Asn Ala Met 375 380 385 390 ctg acg gcg acc ttc tac gcg cag atc aac aac gac tcg atg atc gac 1255 Leu Thr Ala Thr Phe Tyr Ala Gln Ile Asn Asn Asp Ser Met Ile Asp                 395 400 405 gag ctc gtc gac tac gcc cgg ttc tgg cag ccg gac ctg gtg ctc tgg 1303 Glu Leu Val Asp Tyr Ala Arg Phe Trp Gln Pro Asp Leu Val Leu Trp             410 415 420 gag ccc ttc acc ttc gcg ggg gcc atc gcc gcc aag gcg acc ggg gcc 1351 Glu Pro Phe Thr Phe Ala Gly Ala Ile Ala Ala Lys Ala Thr Gly Ala         425 430 435 gct cac gcc cgg ctg ctg gcc ttc ccc gac ctc ttc gcc aac acc cac 1399 Ala His Ala Arg Leu Leu Ala Phe Pro Asp Leu Phe Ala Asn Thr His     440 445 450 ctg acg ctg cgc cgg cgg cag gcg gaa ctg ccc gag gca ctg cgg gac 1447 Leu Thr Leu Arg Arg Arg Gln Ala Glu Leu Pro Glu Ala Leu Arg Asp 455 460 465 470 gac ccg ctg gag gag tgg ctg gcg tac acc ctg gac cgg cac ggg gcc 1495 Asp Pro Leu Glu Glu Trp Leu Ala Tyr Thr Leu Asp Arg His Gly Ala                 475 480 485 gcc ttc gac ccc gac gtg gtc acg gga cag tgg tcg gtc gac cag atg 1543 Ala Phe Asp Pro Asp Val Val Thr Gly Gln Trp Ser Val Asp Gln Met             490 495 500 ccc ccc ggg gtg cgg atg gag ctg ggg ctg ccg acg gtg ccc acg cgc 1591 Pro Pro Gly Val Arg Met Glu Leu Gly Leu Pro Thr Val Pro Thr Arg         505 510 515 tac gtg ccg tac aac ggg ccc ggc ccg gcc gtc gta ccg gac tgg ctg 1639 Tyr Val Pro Tyr Asn Gly Pro Gly Pro Ala Val Val Pro Asp Trp Leu     520 525 530 cgc cgc ccg ccg gag cgc cgc cgg gtc tgc ctc acc ctc ggg ctc acc 1687 Arg Arg Pro Pro Glu Arg Arg Arg Val Cys Leu Thr Leu Gly Leu Thr 535 540 545 550 atc cgg gac acg gag ttc ccc aac gcc gtc gac gtg aac gac gtt ttc 1735 Ile Arg Asp Thr Glu Phe Pro Asn Ala Val Asp Val Asn Asp Val Phe                 555 560 565 gcc tcg ctg gcc gac ctg gac gtc gag gtg gtg gcg acc ctc ggc gag 1783 Ala Ser Leu Ala Asp Leu Asp Val Glu Val Val Ala Thr Leu Gly Glu             570 575 580 aag gaa ctc gcc cag gtg tcg cgg gtg ccg gac aac acg cgg gtg gtc 1831 Lys Glu Leu Ala Gln Val Ser Arg Val Pro Asp Asn Thr Arg Val Val         585 590 595 gac cac gtg gcc ctg ctg gcc ctg ctg ccc agc tgc tcc gcg gtc gtg 1879 Asp His Val Ala Leu Leu Ala Leu Leu Pro Ser Cys Ser Ala Val Val     600 605 610 cac cac ggc ggc gcg ggg acc tgg gcg acc gcc gcc gcc tac ggg gtg 1927 His His Gly Gly Ala Gly Thr Trp Ala Thr Ala Ala Ala Tyr Gly Val 615 620 625 630 ccg cag gtg gcg ctc ggc tgg atg tgg gac gcg atc tac cgg gcc cag 1975 Pro Gln Val Ala Leu Gly Trp Met Trp Asp Ala Ile Tyr Arg Ala Gln                 635 640 645 cgg gtc gag gag ctg ggc gcg gga ctg cat ctg cac tcc gag ggc ctg 2023 Arg Val Glu Glu Leu Gly Ala Gly Leu His Leu His Ser Glu Gly Leu             650 655 660 acc gtc gac atc ctg cgc gac aag ctc gtc cgg gtg ctg gac gac ccg 2071 Thr Val Asp Ile Leu Arg Asp Lys Leu Val Arg Val Leu Asp Asp Pro         665 670 675 gcg ttc acg gag ggg gcg cgg cgg ctg cgc ccg gcc atg ctg tcg gcg 2119 Ala Phe Thr Glu Gly Ala Arg Arg Leu Arg Pro Ala Met Leu Ser Ala     680 685 690 ccg agc ccc aac gaa gtg gtg ccg aca ctg gag aag ttg acc gca cgt 2167 Pro Ser Pro Asn Glu Val Val Pro Thr Leu Glu Lys Leu Thr Ala Arg 695 700 705 710 cac cgg tcg ccg gtc gcg gct cgc tgacgggacg aggacgggga gaagc gtg 2219 His Arg Ser Pro Val Ala Ala Arg Met                 715 aac atc ccg gtg acc ggt gcg gcc ggc ttc atc ggc tcg cac ttc gtc 2267 Asn Ile Pro Val Thr Gly Ala Ala Gly Phe Ile Gly Ser His Phe Val 720 725 730 735 aga aca ctg ctg agc ggc ggt tac ccg ggg cac gag gac gac cgg gtg 2315 Arg Thr Leu Leu Ser Gly Gly Tyr Pro Gly His Glu Asp Asp Arg Val                 740 745 750 acc gtc gtg gac aag ctc acc tac gcg ggg acg ctg aac aac ctg ccc 2363 Thr Val Val Asp Lys Leu Thr Tyr Ala Gly Thr Leu Asn Asn Leu Pro             755 760 765 ccg cgc cat ccg cgg ctg acc ttc gtg cac ggc gac atc tgc gac acc 2411 Pro Arg His Pro Arg Leu Thr Phe Val His Gly Asp Ile Cys Asp Thr         770 775 780 ccg ctg ctg ggc aag gtc ttc ccg ggc cac gag gcg gtg gtg cac ttc 2459 Pro Leu Leu Gly Lys Val Phe Pro Gly His Glu Ala Val Val His Phe     785 790 795 gcc gcg gag tcg cac gtg gac cgt tcg gtg gcg ggt gcc gag gcc ttc 2507 Ala Ala Glu Ser His Val Asp Arg Ser Val Ala Gly Ala Glu Ala Phe 800 805 810 815 gtc cgc acg aac gtg ctc ggc acg cag gcc ctg ctc gaa gcg gcg ctg 2555 Val Arg Thr Asn Val Leu Gly Thr Gln Ala Leu Leu Glu Ala Ala Leu                 820 825 830 cgg cac ggg acg ggc gtc ttc gtc cag gtc tcc acg gac gag acg tac 2603 Arg His Gly Thr Gly Val Phe Val Gln Val Ser Thr Asp Glu Thr Tyr             835 840 845 ggc tcc atc gcc gag gga agc tgg acg gag gac gag ccg ctg ctg ccc 2651 Gly Ser Ile Ala Glu Gly Ser Trp Thr Glu Asp Glu Pro Leu Leu Pro         850 855 860 aac tcc ccg tac gcg gcg tcc aag gcg tcg gcc gat ctg atc gcc cgg 2699 Asn Ser Pro Tyr Ala Ala Ser Lys Ala Ser Ala Asp Leu Ile Ala Arg     865 870 875 tcg tac tgg cgc acc cac ggt ctc gac gtg cgc gtc acc cgg tgc gcc 2747 Ser Tyr Trp Arg Thr His Gly Leu Asp Val Arg Val Thr Arg Cys Ala 880 885 890 895 aac aac tac ggc ccc ggc cag cac ccc gag aag ctg gtc ccg ctg ttc 2795 Asn Asn Tyr Gly Pro Gly Gln His Pro Glu Lys Leu Val Pro Leu Phe                 900 905 910 gtc acc cgg ctc ctc gac ggg cag ccg gtc ccg ctg tac ggg gac ggc 2843 Val Thr Arg Leu Leu Asp Gly Gln Pro Val Pro Leu Tyr Gly Asp Gly             915 920 925 tcg aac ctg cgg gag tgg ctg cac gtc gac gac cac tgc cgg gcg gtg 2891 Ser Asn Leu Arg Glu Trp Leu His Val Asp Asp His Cys Arg Ala Val         930 935 940 cgg ctg gtg ctg gac gag ggc cgg ccg ggg gag atc tac aac atc ggc 2939 Arg Leu Val Leu Asp Glu Gly Arg Pro Gly Glu Ile Tyr Asn Ile Gly     945 950 955 ggc ggc acc cat ctg acc aac aag gag atg acc ggc cgt ctc ctc gcg 2987 Gly Gly Thr His Leu Thr Asn Lys Glu Met Thr Gly Arg Leu Leu Ala 960 965 970 975 ctc tgc ggg cgc gac tgg gac ctg gtc cag cgg gtc gcg gac cgc aag 3035 Leu Cys Gly Arg Asp Trp Asp Leu Val Gln Arg Val Ala Asp Arg Lys                 980 985 990 ggg cac gac ttc cgc tac gcg gtg gac gac tcc aag atc cgc cgc gaa 3083 Gly His Asp Phe Arg Tyr Ala Val Asp Asp Ser Lys Ile Arg Arg Glu             995 1000 1005 ctc ggc tac gcg ccg cgg tgg tcg ctg gag gac gga ctg cgc gag acg 3131 Leu Gly Tyr Ala Pro Arg Trp Ser Leu Glu Asp Gly Leu Arg Glu Thr         1010 1015 1020 gtc gag tgg tac gca gcc cac cgt gac cac tgg gac gcc cgg gag gag 3179 Val Glu Trp Tyr Ala Ala His Arg Asp His Trp Asp Ala Arg Glu Glu     1025 1030 1035 ggc ggc gac ggg ggg tac taaaacttga atatccgggt cgccctcctc 3227 Gly Gly Asp Gly Gly Tyr 1040 1045 tatgctcgcc gggtacccaa atttgagcac ccgaaagaag gggcgggc atg ggc 3281                                                      Met Gly                                                                        ggc gcc gat ttg gcg atc cag acc gaa gct ctg aag aag aaa tac ggc 3329 Gly Ala Asp Leu Ala Ile Gln Thr Glu Ala Leu Lys Lys Lys Tyr Gly         1050 1055 1060 gac aag gag gcc ctc gcg gga ctc gac ctg gag gta ccc cga ggc gtc 3377 Asp Lys Glu Ala Leu Ala Gly Leu Asp Leu Glu Val Pro Arg Gly Val     1065 1070 1075 gta tac ggc ctt ctc ggc ccg aac ggc gcc gga aag acc acc gcg gtc 3425 Val Tyr Gly Leu Leu Gly Pro Asn Gly Ala Gly Lys Thr Thr Ala Val 1080 1085 1090 1095 cgc atc ctc tcc acc ctg ctg aag tac gac gag ggc acc gca cgg gtg 3473 Arg Ile Leu Ser Thr Leu Leu Lys Tyr Asp Glu Gly Thr Ala Arg Val                 1100 1105 1110 gcc ggc tac gac gtc gcc acc cag tcg gcc cag gtc cgc tac agc atc 3521 Ala Gly Tyr Asp Val Ala Thr Gln Ser Ala Gln Val Arg Tyr Ser Ile             1115 1120 1125 gga ctc ctc gga cag cac gcc gcg gtg gac gag gac ctg agc ggc cat 3569 Gly Leu Leu Gly Gln His Ala Ala Val Asp Glu Asp Leu Ser Gly His         1130 1135 1140 cag aac ctc gtg atg ttc ggg cgc tta tac cac atc ggc aag cgc acc 3617 Gln Asn Leu Val Met Phe Gly Arg Leu Tyr His Ile Gly Lys Arg Thr     1145 1150 1155 gcg cag agc cga gcc gac gaa ctg ctg gag cgc ttc ggc ctg acc gaa 3665 Ala Gln Ser Arg Ala Asp Glu Leu Leu Glu Arg Phe Gly Leu Thr Glu 1160 1165 1170 1175 gcc gcc gga aag ccg gcc aag cag tac tcc ggc ggt atg cga cgg cgg 3713 Ala Ala Gly Lys Pro Ala Lys Gln Tyr Ser Gly Gly Met Arg Arg Arg                 1180 1185 1190 ctc gac ctc gcc gcc agt ctc atc ctg gcc ccg tcc gtg ctg ttc ctc 3761 Leu Asp Leu Ala Ala Ser Leu Ile Leu Ala Pro Ser Val Leu Phe Leu             1195 1200 1205 gac gag ccc acc acc ggg ctc gac ccg cgg ggc cgc aac gac gtc tgg 3809 Asp Glu Pro Thr Thr Gly Leu Asp Pro Arg Gly Arg Asn Asp Val Trp         1210 1215 1220 gag tcc gtc cgc ggc ctg gtc tcc gag ggc cgc acg gtc atg ctg acc 3857 Glu Ser Val Arg Gly Leu Val Ser Glu Gly Arg Thr Val Met Leu Thr     1225 1230 1235 acc cag tac ctc gaa gag gcc gac cag ctg gcc gac cgg atc gcc ctc 3905 Thr Gln Tyr Leu Glu Glu Ala Asp Gln Leu Ala Asp Arg Ile Ala Leu 1240 1245 1250 1255 atc gac aac ggc agg gtc gtc gag tcc ggc acc ccg gac cag ctc aag 3953 Ile Asp Asn Gly Arg Val Val Glu Ser Gly Thr Pro Asp Gln Leu Lys                 1260 1265 1270 aac aag atc ggc gga gac cgg atc gac gtc gtg ctc cgc cgc tcc aac 4001 Asn Lys Ile Gly Gly Asp Arg Ile Asp Val Val Leu Arg Arg Ser Asn             1275 1280 1285 gac ctc gaa cgg gcc ggc cgc atg atc ggc gcg ctg ctg cgg gcc gag 4049 Asp Leu Glu Arg Ala Gly Arg Met Ile Gly Ala Leu Leu Arg Ala Glu         1290 1295 1300 ccc gag acg gac acc gac aac cgg cgc gtc agc gtc gtc gtc gcc aac 4097 Pro Glu Thr Asp Thr Asp Asn Arg Arg Val Ser Val Val Val Ala Asn     1305 1310 1315 cgg gtc gcc gcc ctc acc gcg gtc gcc cgg gag ctc 4133 Arg Val Ala Ala Leu Thr Ala Val Ala Arg Glu Leu 1320 1325 1330

[0094] <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: P1 Primer <400> 2 tggtgccgct ggcctgggcg ctgc 24

[0095] <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: P2 Primer <400> 3 ccgccgtggt gcacgaccgc cg 22

[0096] <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: A1 Primer <400> 4 tgatgctctc gaccgcggcc ggctggccgg 30

[0097] <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: A2 Primer <400> 5 ctgcgggcgt cgaacgcgat gctgacggcg 30

[0098] <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: B1 Primer <400> 6 agagcaccag gtccggctgc cagaaccggg 30

[0099] <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: B2 Primer <400> 7 acaacacgcg ggtggtcgac cacgtggccc 30

[0100] <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: R1 Primer <400> 8 cccaagcttg tgcgtgtact gctgaccgc 29

[0101] <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: R2 Primer <400> 9 ccggaattct cagcgagccg cgaccggcg 29

[0102] <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: DS1 Primer <400> 10 cccaagctta tgaaggtgct cgtgacggc 29

[0103] <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> STRANDNESS: single <220> <223> TOPOLOGY: linear <220> <223> Description of Artificial Sequence: DS2 Primer <400> 11 ccggaattcc tagtgccgga cgccctgcc 29

[Brief description of drawings]

FIG. 1 is a diagram showing a method of amplifying the peripheral region of the internal region of rhoG gene by the inverse PCR method.

FIG. 2 is a diagram showing an agarose gel electrophoresis pattern of an amplification product obtained by amplifying the peripheral region of the rhoG gene internal region by the inverse PCR method.

FIG. 3 is a diagram showing a method of constructing a vector pBRG19 for disrupting the rhoG gene.

FIG. 4 TLC of rhoG gene-disrupted strains M7-75 and M7-154
It is a figure which shows an analysis result.

FIG. 5 is a diagram showing the pattern of agarose gel electrophoresis of an amplification product obtained by amplifying a mutation site of a rhoG gene-disrupted strain by PCR.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/10 C12P 19/56 4C086 C12P 19/56 C12R 1:01 // (C12N 1/21 C12N 15 / 00 ZNAA C12R 1:01) (C12N 9/10 C12R 1:01) (C12P 19/56 C12R 1:01) F term (reference) 4B024 AA01 BA10 CA04 DA05 EA04 GA11 4B050 CC03 DD02 LL05 4B064 AF49 CA03 CC24 DA05 4B065 AA50X AA50Y AB01 BA02 CA29 CA44 4C057 AA03 BB02 BB03 CC03 CC04 DD01 JJ51 4C086 EA10 MA01 MA04 NA14 ZB26

Claims (9)

[Claims] 1. A method represented by the following (a), (b) or (c):
An isolated DNA containing a DNA encoding a protein having the ability to transfer rhodosamine to ε-rhodomycinone, or a variant thereof. (a) A DNA consisting of a continuous base sequence from base 914 to base 2191 of SEQ ID NO: 1. (b) a modified form of the DNA shown in (a), which (i) hybridizes with the DNA shown in (a) under stringent conditions, and (ii) ε-rhodomycinone to rhodosamine. DNA encoding a protein having the ability to transfer (c) Due to the degeneracy of gene codons, the DNA shown in (a) above
DNA which does not hybridize under stringent conditions with, but encodes a protein having the same amino acid sequence as the protein encoded by the DNA represented by (a) or (b) above. 2. A protein encoded by the DNA according to claim 1. 3. A recombinant plasmid carrying the DNA according to claim 1. 4. A DNA having a nucleotide sequence in which one or several nucleotides in the nucleotide sequence of claim 1 are substituted, deleted and / or added, and has the ability to transfer rhodosamine to ε-rhodomycinone. DN encoding the deleted protein
Recombinant plasmid carrying A. 5. A transformant lacking the ability to transfer rhodosamine to ε-rhodomycinone, obtained by transforming an actinomycete capable of producing rhodomycin with the recombinant plasmid according to claim 4. 6. An actinomycete lacking the ability to transfer rhodosamine to ε-rhodomycinone, which is obtained by removing a plasmid from the transformant according to claim 5. 7. An actinomycete derived from an actinomycete capable of producing rhodomycin and lacking the ability to transfer rhodosamine to ε-rhodomycinone encodes a protein having the ability to transfer daunosamine to ε-rhodomycinone. Ε-obtained by transformation with a self-replicating or integrating replicative recombinant plasmid carrying the DNA
A transformant having the ability to transfer daunosamine to rhodomycinone. 8. The transformant according to claim 7 is cultured in a medium, and the following formula (I): (However, in the formula, R ₁ is a hydrogen atom or Wherein R ₂ represents a hydrogen atom or a hydroxyl group), and a method for producing the anthracycline antibiotic, characterized in that the anthracycline antibiotic is collected. 9. The following formula (I-2): Anthracycline antibiotic shown by.