KR970002493B1 - Process for preparing adriamycin by fermentation - Google Patents

Abstract

The method for preparing adriamycin has been developed. The adriamycin used as antibiotics is prepared by acquisition of a spore after culturing a Streptomyce peucetius var. caesius of a parent strain in a agar slant medium. Ultraviolet or chemical mutagen on a suspension of this spore is applied to modify it, and then a variant having a tolerance against L-methionine analogue is sorted out, An isolate strain having a high productivity out of them is separated. The isolated strain is cultured in a nutritive medium under an aerobic condition to accumulate the adriamycine, the desired adriamycine is recovered from the culture liquid. 1-32g/L of L-methionine is introduced into the culture medium.

Description

Method for producing adriamycin by fermentation

The present invention relates to a method for preparing the antibiotic adriamycin by a fermentation method, and more particularly, Streptomyces fusethius bar resistant to the L-methionine analog. In the production of adriamycin by culturing Caesius (Streptomyces peucetiusvar.caesius) MWA-72 (KFCC-10798), a method characterized by improving the productivity of adriamycin by adding L-methionine to the medium.

In the biosynthesis process of adriamycin and daunomycin, the anthracycline glycoside antibiotics widely used in the treatment of various tumors, the anthracyclinone moiety is synthesized from acetate and propionate. (J. Am. Chem. Soc. 98, 3370, 1976: Phytochemistey 18, 178, 1979), methoxyl groups are supplied from methionine (Biotechnol. Lett. 1, 471, 1979: J. Antibiot. 31, 178, 1978 The amino sugar portion is known to be derived directly from glucose. (Antibiotiki 21, 299, 1976)

The inventors pay attention to the fact that methionine supplies methoxyl groups in the biosynthetic pathway of adriamycin, streptomyces fusethius bar, a known adriamycin producing strain. Mutation of Kaesius ATCC 27952 to select mutants that are resistant to the el-methionine analogue to adriamycin high production strain Streptomyces fusethius bar. Kaesius MWA-72 (KFCC-10798) has been developed (applicant's patent application filed with the same applicant as the present application: Streptomyces genus microorganisms resistant to the L-methionine analog and a method for producing adriamycin using the same In addition, as a result of investigating the amount of adriamycin accumulated by adding various amino acids to the medium during the culture of the mutant strain, the productivity of adriamycin is improved when L-methionine is added, and the addition time and amount are controlled to about 17%. It has been found that the productivity can be improved and the present invention has been completed.

In other words, an object of the present invention is Streptomyces fusethius bar. A method of culturing Kaesius MWA-72 (KFCC-10798) to accumulate adriamycin and recovering it from the culture to produce admiamycin, wherein fermentation is characterized by adding L-methionine to the culture medium. It is to provide an improved method for producing adriamycin.

Hereinafter, the present invention will be described in more detail.

Parent, Streptomyces fuseotius bar. Streptomyces fusettius bar, which is a variant strain used in the present invention by mutating Kaesius ATCC 27952. The method of obtaining Kaesius MWA-72 (KFCC-10798) can be performed by a normal mutation method. Namely, the parent strain Streptomyces fuseotius bar. After culturing Kaesius ATCC 27952 in agar medium, spores were obtained, and the suspension of the spores was mutated by applying UV or chemical mutagen, and then the mutant strains resistant to the el-methionine analog were selected and doubled adriamycin. It is to isolate a highly productive strain.

The high concentration of adriamycin by culturing the strain according to the method of the present invention can be carried out by adding L-methionine to the culture medium by a conventional method of culturing Streptomyces sp.

The amount of el-methionine added to the medium is in the range of 1-32 g / l, and the addition time may be added at the beginning or during the culture.

As described above, when cultured by adding L-methionine to the medium, the productivity is improved by about 17% compared with the case without addition.

The present invention will be described in more detail with reference to the following Examples, but the present invention is not limited to these Examples.

Reference Example 1

Parent, Streptomyces fuseotius bar. Caesius ATCC 27952 in agar agar medium (20g / l sucrose, 1g / l dry yeast, 2g / l potassium dihydrogen phosphate, 2g / l sodium nitrate, 2g / l magnesium sulfate, 20g / l agar) After culturing for 12 days, the resulting spores were suspended in 10 ml of saline solution, irradiated with ultrasound (Branson 5200) for 30 seconds, and filtered through cotton wool to obtain a spore suspension.

The resulting suspension was washed with brine and then treated with 450 μg / ml of NTG (N-methyl-N-nitro-nitrosoguanidine) at 28 ° C. for 1 hour to cause mutations followed by seleno- as an el-methionine analog. Variants were grown on medium containing about 20 μg / ml methionine and incubated at 28 ° C. for 5-10 days to grow viable strains.

Mutants with resistance to the isolated L-methionine analogs were cultured in the same medium to select high adriamycin strains and passaged in medium in which the concentrations of seleno-methionine were gradually increased to these strains. The mutant strain MWA-72 was screened by screening mutant strains for high concentrations of adriamycin.

Mutant strain Streptomyces fuseotius bar obtained as described above. Kaesius MWA-72 has been deposited with Accession No. KFCC-10798 dated 9 November 1993 to the Korean spawn association.

Example 1

L-cultured for 10-12 days in agar agar medium (20g / l sucrose, 1g / l dry yeast, 2g / l potassium dihydrogen phosphate, 2g / l sodium nitrate, 2g / l magnesium sulfate, 20g / l agar) 50 ml of seed culture medium (glucose 10g / l, peptone 6g / l, yeast extract 5g / l, calcium carbonate 3g / l, magnesium sulfate 0.5g / l, pH 7.0) was added to methionine analogue resistant strain MWA-72 (KFCC-10798). Platinum was inoculated in a 500 ml Erlenmeyer flask containing 2 hours of seed culture at 28 ° C. using a rotary shaker (200 rpm), followed by fermentation broth with various amino acids shown in Table 1 (50 g / sucrose). l, glucose 20 g / l, soybean meal 30 g / l, potassium diphosphate 1 g / l, magnesium sulfate 0.5 g / l, calcium carbonate 4 g / l, ferrous sulfate 0.01 g / l, zinc sulfate 0.01 g / l, 5 ml each of the 500 ml Erlenmeyer flask containing 0.01 g / l copper sulfate, 0.01 g / l cobalt sulfate, and pH 50 ml was inoculated, and main culture was carried out using a white shaker (250 rpm) at 28 ° C. for 7 days. The quantitative analysis of the fermentation broth was performed using HPLC. For pretreatment of the fermentation broth, 2 ml of ethanol was added to 1 ml of the fermentation broth, and the mixture was left at 55 ° C. for 1 hour, followed by centrifugation to obtain a supernatant. Each 10 μl of this solution was analyzed by HPLC under the following conditions, and the amounts of adriamycin produced by the addition of various amino acids are shown in Table 1.

[HPLC Analysis Conditions]

Column: μ-Bondapack C18 (5㎛), 30cm × 3.9cm

Mobile phase: 0.1% phosphate-methanol (25/75)

Flow rate: 1.5ml / min

Detection: UV 254nm

Example 2

It was carried out under the same conditions as in Example 1, but incubated by changing the concentration of L-methionine as shown in Table 2, and measured the amount of adriamycin production is shown in Table 2.

Example 3

It was carried out under the same conditions as in Example 1, but during the incubation of the addition of el-methionine by incubation time to generate adriamycin, the amount of adriamycin produced by HPLC was measured and the results are shown in Table 3.

Claims (3)

Streptomyces fusethius bar resistant to el-methionine analogs. A method of producing adriamycin by aerobically culturing Caesius (Steptomyces peucetius var.caesius) strain MWA-72 (KFCC-10798) in a nutrient medium to accumulate adriamycin and recovering it from the culture medium. A method for producing adriamycin, characterized by participating in L-methionine. The method for preparing adriamycin according to claim 1, wherein the concentration of L-methionine added to the medium is in the range of 1-32 g / l. The method for preparing adriamycin according to claim 1, wherein the L-methionine is added at the beginning or during the culture.