CS259200B1 - Strain of microorganism streptomyces aureofaciens ccm 3978 - Google Patents

Abstract

The present invention relates to a strain of a microorganism Streptomyces aureofaciens CCM 3978, which is characterized by increased production of chlorotetracycline and an increased ratio of chlortetracycline tetracycline, while being resistant against actinophage infection.

Description

The invention relates to a strain of Streptomyces aureofaciens CCM 3978 (internal designation R 8/26) characterized by increased production of chlortetracycline (CTC) and an increased ratio of chlortetracycline to tetracycline (TC), while being resistant to actinophage infection while maintaining stability for many generations.

Increasing the industrial production of the chlorotetracycline antibiotic is possible in two ways: by expanding the production fermentation plants, requiring large investment costs, and by increasing the fermentation yield, which is based either on the higher production capabilities of the strains used for biosynthesis / or better fermentation conditions resulting from improved fermentation control.

One possibility is to find, select or breed a new production strain that would produce chlorotetracycline in higher yields than hitherto as well as in a more favorable ratio to tetracycline, which is produced at the same time, in a conventional plant, in a conventional plant and without interfering with fermentation. In order to expand production without investment costs, breeding of production strains is an important factor. The main indicator of the breeding effect is the increase in the total amount of chlortetracycline per unit volume of fermentation per unit time. The strain must be considered from other points of view; in particular according to the resistance to actinophages, furthermore the ability to grow and sporulate on synthetic and natural substrates, the growth rate of the strain and its resistance to mechanical stress of the mycelium during fermentation.

These objectives were achieved by a new strain of Streptomyces aureofaciens according to the invention, designated R 8/26, deposited in Cs. collection of microorganisms UJEP, Brno, Class of Defenders of Peace 10, under the number CCM 3978

The strain of the invention was obtained from Streptomyces aureofaciens CCM 3766 as follows:

S.aureofaciens CCM 3766 / internal designation 5/211 / i

S.aureofaciens 5 / 211-84

AND

S.aureofaciens R 8/26

The spore suspension of Streptomyces aureofaciens CCM 3766 strain was sown on hardened sporulation medium without selection pressure. Selected colonies were re-plated on sporulated medium agar and tested for production activity as follows:

spore suspension

Even vegetative inoculum

I fermentation

The Streptomyces aureofaciens strain 5 / 211-84 was thus obtained. This strain was cultured in liquid inoculation medium with the addition of phage lysate for 48 hours. The phage-resistant particles obtained were then re-inoculated into the liquid inoculum medium with the addition of the final metabolite (chlortetracycline) at the optimum concentration and cultured for a further 24 hours. Obcultivations were performed on a rotary shaker at 230 rpm, with an eccentric of 25 mm, the culture temperature was 29 ° C.

A sample was collected from the thus obtained population, diluted in distilled water by a decimal row, and sown on solidified non-sporulation medium to obtain isolated colonies. These were visually evaluated after cultivation at 29 ° C for five days, and the selected colonies were inoculated with a metal spatula onto a solidified sporulation medium. The obtained sporulated isolates were further evaluated by fermentation and production levels were determined. Isolates with higher CTC production than the original strain of Streptomyces aureofaciens CCM 3766 were preserved in liquid nitrogen and, after recovery, testo259200

The production-satisfying isolates were further tested for growth and sporulation ability on a natural substrate (peeled pig) and again evaluated for the production of chlortetracycline and the percentage of chlortetracycline and tetracycline. Further, these isolates were assessed for actinophage infection resistance, growth rate, and mycelial resistance to mechanical stress. In this way, a strain according to the invention of Streptomycee au * reofaciens CCM 3978 was obtained.

The Streptomyces aureofaciens CCM 3978 strain forms rounded, wrinkled center colonies on the solidified eporulation medium that pigment dark brown and dark gray sporulate. Strain growth is faster on both stiffened and liquid media than growth of the parent strain Streptomyces aureofaciens CCM 3766. The strain resistance to at least 95% of the actinophage mixture is infected. The strain of the invention produces chlortetracycline in an amount of 10 to 15% greater than that of Streptomyces aureofaciens CCM 3766, and also the percentage of chlortetracycline and tetracycline in the fermentation mixture is much more favorable for the strain of the invention.

Example 1 '

Laboratory scale permentation

Preparation of vegetative inoculum

The vegetative inoculum is prepared in 500 ml boiling flasks with 80 ml of inoculation medium containing the following basic nutrients: sucrose as a carbon source, soy flour, corn steep liquor and ammonium sulphate as nitrogen sources, calcium carbonate and sodium chloride. The medium is inoculated with spores from one third of the surface of the slant agar (possibly with a corresponding amount of spores from the surface of a Petri dish or spores from millet). Cultivation was carried out for 29 hours on a rotary shaker at a rotation speed of 230 per microtiter, with an eccentric of 25 mm. The culture temperature is 29 ° C.

B / Preparation of protected vegetative inoculum

In this method of preparing a vegetative inoculum, the inoculum is protected from unwanted contamination by the final metabo259200

- 4 liters (chlortetracycline). The culture medium is prepared in 500 ml boiling flasks and contains: white dextrin as a carbon source, yeast extract and urea as a nitrogen source, magnesium sulphate and ferrous sulphate and sodium chloride. The flasks are filled with 50 ml of medium to which chlorotetracycline is added at a concentration of 10 to 50 µg / ml. Cultivation is performed for 48 hours on a rotary shaker at 230 rpm, with an eccentric of 25 mm, at 29 ° C. The flasks with the inoculation medium mentioned above are then seeded with 1 ml of the contents of these flasks. In this method of inoculum protection, the production activity of the strain does not decrease, nor does the deterioration of other parameters important in fermentation occur.

C / Production Evaluation of the Phage-Resistant Strain of the Invention Laboratory fermentation "takes place in 500 ml beakers with 40 ml production medium containing sucrose as a carbon source, soybean flour, corn flour, corn steep liquor and sulfuric acid. ammonium wounds as a nitrogen source, calcium carbonate, sodium chloride and other inorganic salts of the necessary trace elements.

At the start of the fermentation, benzylrhodanide is added to the medium at the optimum concentration. Inoculate the flasks with 10% inoculum. Cultivation takes place under the same conditions as for A. Samples for total tetracycline content are taken at 91 and 115 hours of culture. The total content of tetracyclines and chlortetracycline alone is determined by mixing 4 ml of the sample taken in 8 ml of 6% oxalic acid. After standing for 30 minutes, the total tetracycline content is determined by the phosphate method. The ratio of chlortetracycline to tetracycline is determined by paper chromatography.

The production of 5800-6000 .mu.g of chlorotetracycline per ml of laboratory scale medium is achieved as described above. When applied to production scale, the strain of the invention achieves approximately 15,000 µg CTC per ml of medium per 130 culture hours.

Example 2

The preparation of the vegetative inoculum and the fermentation are carried out similarly to Example 1, except that

- 5 large-scale apparatus. The inoculum is prepared in a 5 m tank filled with 3 m inoculum medium. The vegetative inoculum is seeded with a production medium in a 50 nP fermenter filled with 29 m @ 3 of production medium. While maintaining the concentration levels of ammonium ions, sucrose and pH, 12,000 to 14,000 µg of chlorotetracycline per ml of medium is achieved in a conventional fermentation cycle. In the case of an extended fermentation cycle of 130 hours, the production rate kme km not according to the invention proceeds linearly.

Claims (1)

A strain of Streptomyces aureofaciens CCM 3978 with increased production of chlortetracycline and an increased ratio of chlortetracycline to tetracycline while being resistant to actinophage infection.