KR100266435B1 - Mutant strains producing anthracyclin system antibiotics and process for preparing doxorubicin with mixed culture - Google Patents

Abstract

The present invention relates to a mutant strain producing anthracycline-based antibiotics and a method for mass production of doxorubicin by mixing and culturing the same. Specifically, in the present invention, a mutation is induced during the biosynthesis process of doxorubicin, which does not produce doxorubicin, but the activities of the enzymes necessary for the biosynthesis of intermediates after the biosynthetic intermediate ε-rhodomycinone retain Streptomyces fus Mutations in the biosynthesis process of the Setius mutant strain, baumycin, resulted in a high yield of the intermediate ε-rhodomycinone, a strain of the genus Streptomyces, a mixture of the strains and their transformants It relates to a fermentation method of culturing to produce doxorubicin, by using the method can be easily purified by mass production of doxorubicin excellent anti-cancer effect.

Description

Mutant strains producing anthracyclin system antibiotics and process for preparing doxorubicin with mixed culture}

The present invention relates to a mutant strain producing an anthracycline antibiotic and a method of mass production of doxorubicin (aka: adriamycin) and derivatives thereof by mixing and culturing the same.

More specifically, the present invention does not produce doxorubicin due to mutations in the biosynthesis process of doxorubicin, but does not produce doxorubicin, but retains the activity of the enzymes necessary for the biosynthesis of intermediates after the biosynthetic intermediate ε-rhodomycinone. Mutations in the biosynthesis process of the Seth fusetius mutant strain, Baumycin, result in a high yield of intermediate ε-rhodomycinone, the strains and their transformants It relates to a fermentation method of mass production of doxorubicin by mixing and culturing.

Anthracycline antibiotics are compounds with a 7,8,9,10-tetrahydro-5,12-naphthacequinone backbone, which have been reported to be effective in treating breast cancer, bladder cancer, Hodgkin's disease and leukemia. It is widely used as an anticancer agent around the world. Among these, daunorubicin (aka daunomycin) was first introduced in 1963 by Streptomyces peucetius ATCC 29050; US Pat . No. 3,092,550; Int. J. Syst. Bact. 30 : 396, 1980) and doxorubicin was first isolated in 1969 from another variant strain, Streptomyces peucetius subsp.caesius ATCC 27952 (US Pat. No. 3,590,028). Ho., Arcamone, F et al ., Biotech, Bioengineer., 11: 1101-1110, 1969).

Such doxorubicin is most widely used as a chemotherapy treatment with excellent anticancer effects, but there are still many difficulties in mass production due to its low efficiency and difficulty in purification.

Until now, the production process of doxorubicin is not a method of directly fermenting doxorubicin, but a method of producing and separating daunorubicin, which has a high yield of production, using microorganisms first by fermentation, separating it, and then transferring it to a chemical reaction process to substitute doxorubicin. Has been used primarily (US Pat. No. 3,803,124, 1974). Therefore, the process is not only complicated and cumbersome, but also expensive to produce doxorubicin.

Therefore, the present inventors continued research to develop a method for mass production of doxorubicin, which is useful as an anticancer agent. As a result, Streptomyces genus Asus Kaesius strain producing Doxorubicin and Streptomyces sp. Strain C5 producing Baumycin Mutation strains were induced at high yields of mutant strains possessing only the enzyme activity necessary for biosynthesis after acrobinone, a biosynthetic intermediate during doxorubicin, and ε-rhodomycinone, a biosynthetic intermediate during biosynthesis of bamycin. The present invention was completed by developing a method for obtaining a mutant strain to be produced and efficiently producing doxorubicin by mixing and culturing the mutant strain under appropriate conditions.

It is an object of the present invention to provide a method for mass production of doxorubicin by mixing and culturing a mutant strain having a similar biosynthesis process for producing an anthracycline antibiotic.

In order to achieve the above object, the present invention is mixed with the mutant strains that produce mutations in the biosynthesis process of the anthracycline antibiotics to produce only biosynthetic intermediates and mutant strains retaining the activity of the biosynthetic enzymes after the intermediate doxorubicin and Provided are methods for producing derivatives thereof.

Specifically, the present invention is a mutant strain producing a high yield of the biosynthetic intermediate ε-rhodomycinone as a mutant strain and a mutant strain having a biosynthetic enzyme activity after ε-rhodomycinone while blocking the biosynthetic process of doxorubicin Use At this time, the transformants transformed with the doxorubicin resistance gene drr AB and the mutant strains which produced the ε-rhodomycinone in high yield and the doxorubicin C-14 hydroxylase gene were blocked. Transformants transformed with dox A can be used.

In addition, the present invention provides a Streptomyces fusettius mutant strain Nu23 (Accession No .: KCTC 0382 BP), which has a biosynthetic enzyme activity after acrobinone prepared by a mutation process and is blocked in the biosynthesis process of doxorubicin.

In addition, the present invention can provide an expression vector capable of expressing the daunorubicin C-14 hydroxylase gene dox A and a Streptomyces fusettius variant strain Nu23 (dox A +) transformed with the expression vector. .

In addition, the present invention provides a strain Streptomyces genus strain D5 (Accession No .: KCTC 0383 BP), which produces ε-rhodomycinone produced by a high yield in a high yield.

In addition, the present invention can provide an expression vector capable of expressing the doxorubicin resistance gene drr AB and a strain strain D5 (drr AB +) of Streptomyces transformed with the expression vector.

In addition, the present invention uses a 13-dihydrodaunorubicin as a substrate and the expression vector containing the dox A gene or a strain expressing a large amount of daunorubicin C-14 hydroxylase or the enzyme directly or appropriate Provided is a method for mass production of doxorubicin using an enzyme attached to a carrier.

Hereinafter, the present invention will be described in detail.

The present invention produces a doxorubicin (aka adriamycin) and derivatives thereof useful as an anticancer agent by mixing and culturing strains having a similar biosynthesis process to produce anthracycline-based antibiotics and mutating the same.

Specifically, doxorubicin is produced using a mutant strain producing only the intermediate of the biosynthetic process and a mutant strain showing only the activity of the biosynthetic enzyme after the intermediate.

For reference, as shown in Figure 1 , the anthracycline-based antibiotics doxorubicin and baumycin (aklavinone), ε-rhodomycinone (ε-rhodomycinone), daunorubicin (daunorubicin) in the microorganisms It is synthesize | combined via intermediates, such as these.

First, the present invention doxorubicin and daunorubicin: causes an artificial mutation by using a (also known as daunomycin, daunomycin) producing Streptomyces Fu Shetty mouse subgenus siwooseu Car (Streptomyces peucetius subsp caesius.) Strain in which. Specifically, the biosynthesis of the streptomyces fusettius subgenus Kaesius ATCC 27952 strain by artificial mutagenesis by the general procedure using ultraviolet light and MNNG does not produce doxorubicin but is an intermediate of the biosynthesis process. Enzymatic activity produces variant strains that retain.

The mutant strain was named Streptomyces Pusetius subgenus Kaesius Nu23 strain and was deposited on September 8, 1997 to the Genetic Bank of Korea Institute of Science and Technology (accession number: KCTC 0382 BP).

The invention also relates to Streptomyces sp. C5 strain (Padney, RC, Proc. Biochem., 13: 6-13, 1979, Bartel, PL, J. Gen. Microbiol. , 136: 1877-1886, 1990), but mutant strains are produced by high yields of ε-rhodomycinone, which does not produce Baumycin but is an intermediate of its biosynthetic process.

The mutant strain was named D5 strain of the genus Streptomyces and was deposited on September 8, 1997 to the Genetic Bank of Korea Research Institute of Science and Technology (accession number: KCTC 0383 BP).

In order to select the artificial mutant strains, strains of Streptomyces genus are cultured using R2YE medium and NDYE medium, and by adding mutagen. The characteristics of the selected strains were examined by the growth and color of parasitic mycelia of each strain, the growth and color of the substrate mycelia, the color of the back side of the medium, the production of water-soluble pigments, and the like.

In addition, the present invention is to analyze the characteristics of the mutation by analyzing doxorubicin and its biosynthetic intermediates produced by the mutant strains, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), etc. Was performed and these compounds were quantitated.

Specifically, the mutant strain Nu23 of the present invention was cultured by adding acrobinone, ε-rhodomycinone, daunorubicin, 13-dihydrodaunorubicin, and the like to the medium, respectively, and these compounds were each in an appropriate ratio. It was confirmed that the mutant strain retained the enzyme activity involved in these biosynthetic processes without producing the biosynthetic intermediates directly.

In addition, the mutant strain D5 of the present invention produces ε-rhodomycinone at a high concentration until the 6th day of culture, and its concentration decreases from the 7th day, there is no resistance to daunorubicin, and 40 μg / ml also for doxorubicin. It was confirmed that there was no resistance above the concentration.

In the present invention, the mutant strain D5 and the mutant strain D5 producing high yields of the biosynthetic intermediate of anthracycline antibiotics have the biosynthetic enzyme activity after ε-rhodomycinone, but the biosynthetic process of doxorubicin is blocked. Nu23 is mixed and cultured to mass produce doxorubicin.

At this time, mutant strain having biosynthetic enzyme activity after ε-rhodomycinone and blocking the doxorubicin biosynthesis process, Daunorubicin C-14, an important enzyme that acts as a catalyst for the conversion of daunorubicin to doxorubicin. It is more preferable to use the thing which expressed the large amount of hydroxylase gene dox A.

The present invention may use a transformant Nu23 (dox A +) which prepares an expression vector including the dox A gene and transforms it into the mutant strain.

In addition, it is more preferable to use a mutant strain that produces ε-rhodomycinone in a high yield to enhance resistance to doxorubicin and continue to produce doxorubicin and its derivatives.

The present invention can use the transformant D5 (drr AB +) to prepare an expression vector comprising the doxorubicin resistance gene drr AB and transformed it to the above-mentioned strain.

The present invention produces a large amount of doxorubicin and its derivatives by fermentation while controlling the mixing time and mixing amount of each culture medium using the above mutant strains. At this time, the mutant produces ε-rhodomycinone in high yield. As a strain, it is preferable to use a culture medium at a time when the yield of ε-rhodomycinone is the highest, and as a strain having a biosynthetic enzyme activity after ε-rhodomycinone, a culture medium suitably cultured is used, and these strains are mixed. Incubate by

Specifically, in the case of the mutant strain D5, it is preferable to use the culture solution on the 6th day having the highest epsilon-rhodomycinone production, and in the case of the mutant strain Nu23, it is preferable to use the culture medium cultured for about 3 days.

In addition, centrifugation of the culture of the mutant strain Nu23 followed by mixed culture minimizes the effects of biosynthetic regulators or culture volumes that may exist in the culture, but more than three times more production of daunorubicin inhibits the survival of the mutant strain D5. This can radically block the production of ε-rhodomycinone.

Thus, as described above, the recombinant variant strain D5 (drr AB +) expressing the drr AB (d.H. Youngbi et al ., J. Microbiol, Biotech., 2: 153-160, 1992), which is a doxorubicin resistance gene, is present in the variant strain D5. By using it, the resistance to doxorubicin or daunorubicin produced during fermentation increases, and the strain continues to proliferate, and thus it is possible to continuously supply ε-rhodomycinone, an intermediate of the doxorubicin biosynthesis process, and the mixed culture method of the present invention. It is possible to improve the productivity of doxorubicin, the final target compound in. In addition, it is possible to solve the problem that the separation and purification process is complicated by the remaining of daunorubicin that is not converted to doxorubicin.

In addition, the recombinant strain Nu23 expressing the daunorubicin C-14 hydroxylase gene dox A (Dickens, ML & Strohl WR, J. Bacteriol., 178: 3389-3395, 1996) on the mutant strain Nu23. ) Is mixed with other recombinant strain D5 (drr AB +), which will facilitate the supply of ε-rhodomycinone, an intermediate of the doxorubicin biosynthesis process, and effectively convert the remaining daunorubicin in the culture medium into doxorubicin. The productivity of doxorubicin can be significantly increased and the purification process can be made simpler.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

The following examples are only for illustrating the present invention, but the content of the present invention is not limited thereto.

Example 1 Production of Streptomyces Fusethius Subgenus Kaesius Mutant Strains Induced Mutations in Doxorubicin Biosynthesis

In the present invention, mutations are induced in an anthracycline-based antibiotic doxorubicin biosynthesis process, but do not produce doxorubicin, but the biosynthetic enzymes after the biosynthetic intermediate acrobinone are mutant strains to produce an active mutant strain. Setius subspae Caesius ATCC 27592 strain was selected and cultured in NDYE medium (composition described below). At this time, the strain produced 10-15 μg / ml of ε-rhodomycinone, 3-5 μg / ml of daunorubicin, and 0.5-1 μg / ml of doxorubicin.

The spores of the strain were then suspended in TM buffer (TM: 50 mM Tris-hydrochloric acid, 50 mM maleate, pH 8.5) and the mutant derivative MNNG (MNNG: N-methyl-N'-nitro-N-nitrosoguanidine) The final concentration was added to 1 mg / ml and treated for about 40 minutes. Next, the viable colonies were selected by culturing in R2YE solid medium (composition described below) to which doxorubicin was added at a concentration of 300 μg / ml. As a result, a mutant strain N13 was produced that produced doxorubicin at a concentration of 8-10 μg / ml, and the mutant strain N13 was irradiated with ultraviolet light for 20-60 seconds for mutagenicity, and then mutant strain Nu23 having a purple spore was selected.

Doxorubicin and its biosynthetic intermediates in its metabolites were examined using the mutant strain Nu23 by the method of the following reference example, which is not only doxorubicin but also intermediates of the doxorubicin biosynthesis process, daunorubicin, ε-rhodomycinone and aclabinone (aklavinone) did not produce. However, the intermediates of doxorubicin biosynthesis, daunorubicin, ε-rhodomycinone, acrobinone and 13-dihydrodaunorubicin (13-dihydrodaunorubicin) were added to the liquid medium in which the mutant strain Nu23 was incubated for 3 days, respectively. Continued culture confirmed that doxorubicin is produced again. Although the biosynthesis of the mutant strain Nu23 up to acrobinone, which is an intermediate of the doxorubicin biosynthesis process, is blocked, the final targets of daunorubicin and doxorubicin are the final targets when acrobinone, ε-rhodomycinone, and daunorubicin are added as substrates. It is to confirm that it is a mutant strain that fully retains the enzyme activity of the conversion process that can produce such.

Streptomyces fusettius Nu23 strain mutated in the doxorubicin biosynthesis process of the present invention was deposited on September 8, 1997 to the Gene Technology Bank of Korea Institute of Science and Technology (Accession Number: KCTC 0382 BP).

Example 2 Production of Streptomyces spp. Strains Producing High Yield of ε-Rhodomycinone

In the present invention, in order to produce a mutant strain producing a high yield of the biosynthetic intermediate ε-rhodomycinone during the biosynthesis process of the anthracycline-based antibiotic Baumycin, C5 strain of Streptomyces as a parent strain ( Streptomyces sp. C5; Padney, RC, Proc. Biochem., 13: 6-13, 1979; Bartel, PL, J. Gen. Microbiol., 136: 1877-1886, 1990). Spores of C5 genus C5 (10 ⁹ cfu / ml) were suspended in TM buffer (TM: 1 M Tris-hydrochloric acid, 50 mM maleate, pH 8.5), and the mutant derivative MNNG (MNNG) was finalized. The concentration was added to 1 mg / ml and treated at 37 ° C. for 50 minutes. Next, the mutant strain D5, which was colonized with spores of different colors, was selected from the colonies surviving for 7 days by plating in R2YE medium.

As a result of analyzing the metabolites produced by the method of the following reference example using the mutant strain D5, the mutant strain 166μg / rho-myinone when cultured for 6 days in NDYE medium without producing baumycin It was confirmed to produce to ml concentration. In the same manner as the production of the metabolite, the cell mass was increased to the concentration of 15 g / L until the 6th day of culture, and then began to decrease from the 7th day. During this period, the pH was little changed between 7.5 and 8.5, but gradually increased. It could be confirmed (see FIG. 2 ).

Streptomyces sp. D5, which produces ε-rhodomycinone of the present invention in high yield, was deposited on September 8, 1997, to the Genetic Bank of Korea Institute of Science and Technology (Accession No .: KCTC 0383 BP). .

The composition of R2YE medium and NDYE medium used for the selection of the variant strains is as follows. In each medium, the liquid medium was prepared without adding agar, and the solid medium was used by adding only agar at a concentration of 1.5% in the same composition.

(1) R2YE medium

103 g of sugar

K ₂ SO ₄ 0.25 g

MgCl ₂ · 6H ₂ O 10 g

0.1 g of casamino acid

5.0 g of yeast extract

TES (N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid)

                                            5.73 g

790 ml of distilled water

Dissolve and sterilize, and 5 ml of 1 M NaOH, 10 ml of 0.5% KH ₂ PO ₄ , 20 ml of 1 M CaCl _2, 15 ml of 20% proline, and 2 ml of trace elements Add to use. The trace elements used at this time were ZnCl ₂ 40 mg, FeCl ₃ · 6H ₂ O 200 mg, CuCl ₂ · 2H ₂ O 10 mg, MnCl ₂ · 4H ₂ O 10 mg, Na ₂ B ₄ O ₇ · 10H ₂ O 10 mg, (HN ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O 10 mg, which are prepared by dissolving them in 1 L of distilled water.

(2) NDYE badge

Maltose 45 g

NaNO ₃ 4.28 g

K ₂ HPO ₄ 3 H ₂ O 0.18 g

MgSO ₄ 7H ₂ O 0.12 g

5 g of yeast extract

5.22 g of hydroxyethyl piperazine ethanesulfonic acid (HEPES)

2 ml of trace elements are dissolved in distilled water, adjusted to pH 7.2, and the final volume is prepared to 1 L and sterilized.

Example 3 Culture Characteristics of Mutant Strains of the Invention

In order to investigate the culture characteristics of the mutant strains selected in the above examples, these strains were cultivated at 28 ° C. for 14 days using various media to confirm their characteristics. Specifically, the growth and color of the parasitic mycelia of each strain, the growth and color of the substrate mycelia, the color of the back side of the medium, the culture characteristics were investigated by focusing on the generation of water-soluble pigments and the like are shown in Table 1.

Comparison of Morphological Characteristics of Mutant Strains Type of badge Type of strain Growth and Color of Parasitic Mycelia Growth and color of substrate mycelia Color on the back of the badge Water soluble pigment Growth color Growth color Oatmeal Agar (ISP3) 27952 usually Light yellow Good Light yellow Light yellow radish Nu23 Normal, no spores yellow Good yellow Yellow red radish C5 Good White gray Good Dark yellow Yellow red radish D5 Good White gray Good maroon maroon radish NDYE 27952 Good Dark skin Good Light yellow High color radish Nu23 Normal, no spores Light yellow Good Light yellow Light yellow radish C5 Good White Good Brown Brown radish D5 Good White Good Brown Brown radish R2YE 27952 usually Yellowish white usually Light white Light white radish Nu23 Normal, no spores Light white usually Light white Light white radish C5 Good White usually maroon maroon radish D5 Good White usually maroon maroon radish Yeast Starch Agar (YS) 27952 usually Dark gray usually Light yellow Light yellow radish Nu23 Normal, no spores Light yellow usually Light yellow Light yellow radish C5 usually grey usually Light yellow Light yellow radish D5 usually grey usually Light yellow Light yellow radish

Reference Example Method for Quantifying Doxorubicin and its Biosynthetic Intermediates

In the present invention, in order to analyze doxorubicin and its biosynthetic intermediates produced by the mutant strains, they were quantified using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

The mutant strain Nu23 and mutant strain D5 were shaken in culture (250-300 rpm) for 7-8 days in a range of temperature 27-32 ° C. under aerobic conditions using NDYE medium. Oxalic acid (oxalic acid) was added to the culture solution at a concentration of 30 mg / ml, hydrolyzed at 55 ° C. for 45 minutes, and the pH of the medium was adjusted to 8.5 using 10 N NaOH. The reaction mixture was mixed well by adding the same amount of chloroform: methanol mixed solvent mixed in a 9: 1 ratio, and then extracted with a solvent layer and centrifuged at 3,000 rpm (revolution per minute) for 30 minutes. The solvent layer was recovered and concentrated under reduced pressure. It was again dissolved in a small amount of methanol and analyzed by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC).

In this case, in the case of TLC analysis, a developing solvent for analyzing a biosynthetic intermediate in a sugar-coated state is a mixture of chloroform: methanol: formic acid in an 80: 20: 2 ratio. As a developing solvent for analyzing this unbound biosynthetic intermediate, a mixture of hexane, chloroform, and methanol in a 5: 5: 1 ratio was used. As a plate used for the TLC analysis, a silica gel 60 F-254 manufactured by Merck was used.

In the case of HPLC analysis, an ODS-120T C-18 column manufactured by Tosoh Co., Ltd. was used, and as a developing solvent, acetonitrile: 20 mM phosphate solution (acetonitrile: Sodium dodecyl sulfate (SDS) was added at a final concentration of 10 mM in a solvent mixed with 20 mM phosphoric acid in a 42:58 ratio.

Example 3 Investigation of Metabolic Properties of Streptomyces Fusethius Strain Nu23

In order to analyze the mutation characteristics of the doxorubicin biosynthesis process in the mutant strain Nu23 of the present invention, the mutant strain Nu23 was incubated at 28 ° C. for 50 days in 50 ml of NDYE liquid medium in a 250 ml flask. Next, each of the biosynthetic intermediates was further incubated for 72 hours by adding 10 μg / ml of acrobinone, ε-rhodomycinone, daunorubicin, and 13-dihydrodaunorubicin to the medium. And doxorubicin were measured in amounts.

As a result, as shown in Table 2, acrobinone was converted to doxorubicin by 35% and ε-rhodobycinone was converted by 49%. However, about 16% and 34% of daunorubicin and 13-dihydrodaunorubicin were converted to doxorubicin, respectively. Of particular interest is the fact that 13-dihydrodaunorubicin has a higher conversion rate than daunorubicin, and that 13-dihydrodaunorubicin is the last enzyme in the doxorubicin C-14 hydroxylase, the final enzyme in the doxorubicin biosynthesis process. It suggests the possibility of temperament. In addition, it is obvious to those skilled in the art that the conversion rate of the intermediates may vary according to the change of the medium or the change of culture conditions, but this variant strain is involved in the conversion process without producing these intermediates. Retaining enzyme activity is important.

Conversion Rate of Intermediates in Mutant Strain Nu23 Added intermediates Concentration ¹ (μg / ml) Conversion ε-rhodomycinone Daurorubicin 13-dihydrodaurorubicin Doxorubicin Accrabinon ^** 10 5% 25% 30% 35% ε-rhodomycinone 10 9% ^* 20% 22% 49% 13-dihydrodaurorubicin 10 - 25% 41% ^* 34% Daurorubicin 10 - 45% ^* 39% 16% ¹ : Final concentration of intermediate added to medium incubated for 3 days in NDYE liquid medium ^* : Percentage of intermediate remaining after 72 hours of incubation with intermediate added ^** : Accrabinone remains below 5% after addition

Example 4 Investigation of Metabolic Characteristics of Streptomyces Strain D5

In order to analyze the mutation characteristics of the Baumycin biosynthesis process of the mutant strain D5 of the present invention, the spores of the mutant strain D5 were inoculated into a 250 ml flask with 50 ml of NDYE liquid medium and incubated at 28 ° C. for 3 days. Each biosynthetic intermediate and the like were analyzed by the reference example. As a result, the mutant strain D5 produced ε-rhodomycinone up to a concentration of 166 μg / ml on the 6th day of culture, and the concentration was gradually decreased from the 7th day. It was confirmed that it increased to the concentration of 15 g / L on day 1 and began to decrease from day 7. During this period, the change in pH was found to increase gradually, with little change between 7.5 and 8.5.

In addition, in order to confirm resistance to the final product doxorubicin and its biosynthetic intermediate daunorubicin using the mutant strain D5, doxorubicin and daunorubicin are added at concentrations of 10, 20, 30, 40, and 50 μg / ml, respectively. Spores (1.3 × 10 ⁸ cfu / ml) of the mutant strain D5 were smeared on the prepared R2YE solid medium and cultured at 28 ° C. for 5 days, and the number of colonies surviving was measured. As a result, as shown in Table 3, the mutant strain D5 did not show any resistance to daunorubicin, and did not show resistance to doxorubicin at a concentration of 40 μg / ml or more.

Drug Resistance of Mutant Strain D5 to Daunorubicin and Doxorubicin          Anticancer concentration (μg / ml) Daunorubicin Doxorubicin Survival Colony Count % Survival Colony Count % 0 μg / ml 1.3 × 10 ⁸ 100 1.3 × 10 ⁸ 100 10 μg / ml 0 0 0.4 × 10 ⁸ 31 20 μg / ml 0 0 0.2 × 10 ⁸ 17 30 μg / ml 0 0 0.06 × 10 ⁸ 4.5 40 μg / ml 0 0 0.02 × 10 ⁸ 1.5 50 μg / ml 0 0 0 0

Example 5 Mixed Culture Method of Mutant Strain Producing Doxorubicin

In the present invention, in order to mass produce doxorubicin, 100 ml of NDYE liquid medium is inoculated into 500 ml baffled flasks of the mutant strain D5 and the mutant strain Nu23, respectively, incubated at 28 ° C., and then mixed with each culture solution. Fermentation was again carried out under three different conditions by adjusting the time and amount of mixing.

First, the culture medium was mixed at the same volume as the culture solution of the mutant strain Nu23 cultured for 3 days, on the third day at which the mutant strain D5 started producing ε-rhodomycinone. As a result of performing a mixed culture of type A in which the culture medium of the mutant strain Nu23, which had been cultured for three days, was cultured on the third day of the mutant strain D5 starting to produce 20 μg / ml of ε-rhodomycinone, and then mixed. On day 1, 2 μg / ml of daurorubicin was confirmed, and on day 2 of the mixing, trace amounts of doxorubicin were also observed. On the third day, doxorubicin was observed at about 17 μg / ml, and on the fourth day of mixing, most 8 μg / ml of daunorubicin was synthesized. Therefore, it was confirmed that the biosynthesis reaction after ε-rhodomycinone proceeded by using the ε-rhodomycinone produced by the mutant strain D5 by the mixed culture of the two strains in the mutant strain Nu23. However, the amount of epsilon-rhodomycinone production in the initial stage of mixed culture did not increase in the same manner as when culturing the mutant strain D5 alone, and doxorubicin was not produced properly (see FIG. 3 ).

Example 6 Mixed Culture Method of Mutant Strain Producing Doxorubicin

In order to mass produce doxorubicin, the present invention is inoculated with 100 ml of NDYE liquid medium in 500 ml baffled flasks of the mutant strain D5 and mutant strain Nu23, respectively, and incubated at 28 ° C., followed by ε in mutant strain D5. On day 6, the highest amount of rhodomycinone production and the culture solution of mutant strain Nu23 cultured for 3 days were mixed and cultured in the same volume.

As a result, a mixed culture of type B was carried out by mixing the culture medium of the mutant strain D5 having the highest yield of ε-rhodomycinone with the same volume of the culture medium of the mutant strain Nu23 cultured for three days. Doxorubicin was produced in the culture medium at 104 μg / ml, and daunorubicin was produced up to 33 μg / ml on the first day of mixing. In this case, more than 90% of the ε-rhodomycinone produced by the mutant strain D5 was observed to be converted to daunorubicin and doxorubicin (see FIG. 4 ). This suggests the possibility of producing more products in mixed culture with variant strains with higher substrate productivity.

Example 7 Mixed Culture Method of Mutant Strain Producing Doxorubicin

In order to mass-produce doxorubicin, the present invention was inoculated with 100 ml of NDYE liquid medium in 500 ml baffled flasks of the mutant strain D5 and mutant strain Nu23, respectively, and incubated at 28 ° C. for 3 days. Variant strain Nu23 was centrifuged to add only cells to the three-day culture or six-day culture of variant strain D5. This is to minimize the effects of biosynthetic regulators or changes in culture volume that may be present in the culture of the mutant strain Nu23.

As a result of the mixed culture, a very small amount of daurorubicin and doxorubicin was produced in the three-day culture medium of the mutant strain D5 as shown in FIG. 3, and up to 97 μg / ml of daunorubicin was produced in the six-day culture medium. Doxorubicin produced about 47 μg / ml. This indicates that the continuous supply of the intermediate epsilon-rhodomycinone is an important factor in the production of doxorubicin in the mixed culture conditions of the present invention. In addition, it was confirmed that 80 μg / ml of daunorubicin was produced in the 6-day culture of the mutant strain D5 three times more than the mixed culture of type B after 2 days of mixed culture (see FIG. 5). However, this excessive production of daunorubicin inhibits the survival of the mutant strain D5 strain, which not only rapidly blocks the production of ε-rhodomycinone, but also causes the excess daunorubicin to accumulate without rapid conversion to doxorubicin.

As described above, the fermentation method of the present invention by producing a mixed culture of mutant strains producing anthracycline-based antibiotics efficiently to produce doxorubicin, so far has excellent anti-cancer effect, but its efficiency is low and the purification process is difficult. Doxorubicin that has come can be obtained easily and economically in large quantities.

Figure 1 shows the biosynthesis process of doxorubicin and baumycin of the present invention,

Figure 2 shows the increase in the production of ε- rhodomycin with the growth of Streptomyces genus strain D5 of the present invention,

○: pH; ▽: dry cell weight; ◆: ε-rhodomycinone

Figure 3 shows the culture of the culturing the strain strain D5 culture for 3 days and the culture medium cultured Streptomyces Pusetius mutant strain Nu23 for 3 days to produce a doxorubicin, an anticancer agent,

◇: ε-rhodomycinone; ●: doxorubicin; ■: Daunorubicin

Figure 4 shows the culture of the culture medium cultured the mutant strain D5 culture for 6 days and the culture medium cultured the mutant strain Nu23 for 3 days mixed culture of doxorubicin is produced,

Figure 5 shows the culture of the culture medium obtained by centrifugation of the culture medium in which the mutant strain D5 was cultured for 6 days and the culture medium in which the mutant strain Nu23 was cultured for 3 days, the doxorubicin is produced as an anticancer agent.

Claims (6)

Streptomyces fusettius mutant strain Nu23 (accession number: KCTC 0382 BP), which has a biosynthetic enzyme activity after acrobinone, a biosynthetic intermediate produced by the mutation process, and has blocked the biosynthetic process of doxorubicin. The Streptomyces fusetius mutant strain Nu23 according to claim 1, wherein the variant strain Nu23 may comprise an expression vector expressing the daunorubicin C-14 hydroxylase gene dox A. Mutant strain D5 genus Streptomyces producing high yield of ε-rhodomycinone prepared by the mutation process (Accession No .: KCTC 0383 BP). The variant strain of Streptomyces genus D5 according to claim 3, wherein the variant strain D5 may comprise an expression vector capable of expressing the doxorubicin resistance gene drr AB . A method for producing doxorubicin using 13-dihydrodaunorubicin as a substrate using the mutant strain of claim 2. Iii) culturing the mutant strain Nu23 having the biosynthetic enzyme activity after ε-rhodomycinone of claim 1 and blocking the biosynthetic process of doxorubicin; Ii) culturing the mutant strain D5 to produce ε-rhodomycinone, the biosynthetic intermediate of claim 3, in high yield; Iii) adding the mutant strain Nu23 cultured in step iii) to the culture solution of step ii); And Iii) a method for mass production of doxorubicin and its derivatives comprising the step of culturing the mutant strains Nu23 and D5

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