HU209929B - Process for production of thiostrepton antibiotic process for production of thiostrepton antibiotic - Google Patents

Abstract

The present invention relates to a method for the preparation of a thiostrepton antibiotic by means of microbiology, aerobic fermentation, carbon and nitrogen source assimilable with a strain of Streptomyces laurentii, and mineral salts, by culturing the chloramphenicol resistant mutant of the strain Streptomyces laurentii as a microorganism and after fermentation. the resulting antibiotic is isolated from the cells. EN 209 929 B Description: 6 pages

Description

The present invention relates to a process for the preparation of an antibiotic thiostrepton by microbial digestion by aerobic fermentation, a chloramphenicol mutant of a strain of a microorganism of the species Streptomyces laurentii, a medium containing assimilable carbon and nitrogen sources and mineral salts.

Thiostrepton is a cyclic polypeptide antibiotic produced by strains of microorganisms belonging to various species of Streptomyces, such as strains of Streptomyces azureus, hawaiiensis and laurentii. This property of each Streptomyces species was first described in U.S. Patent No. 2,982,689. U.S. Pat. Accordingly, the strain of the species Streptomyces laurentii is cultivated on an industrial scale under aerobic conditions in a medium containing assimilable carbon and nitrogen sources and mineral salts, and the antibiotic formed during fermentation is isolated from the fermentation broth.

Hitherto known processes for the preparation of thiostrepton have a relatively low yield. Although a process has been described in which thiostrepton is produced in large quantities in the fermentation broth, the process can only be achieved by continuous fermentation using sophisticated control equipment under laboratory conditions, so that this scientific result has not yet been commercially applied.

In the production of industrial-scale thiostrepton, the fermentation is carried out intermittently for 4-5 days with aerobic fermentation, and the concentration of thiostrepton in the fermentation broth is 1000 to 100 pg / ml / Appl. Microbiol. Biotechnol. 25: 520531 (1987). The aim of our experiments was to increase the efficiency of the process by isolation of new, more productive strains in strains of the strain of Streptomyces laurentii. It is known that aerial mycelium formation associated with spore formation and antibiotic production are co-regulated in most species of the Streptomyces genus / Doull J.L. and Vining L.C., Biotech. Adv. 8, 141-158 (1990). While many details of this regulation are unclear, several experimental data show that it is related to the intracellular synthesis of guanosine triphosphate (GTP) and guanosine tetra- and pentaphosphate (ppGpp and pppGpp). Ochi, K., J. Bact. 169: 3608-3616 (1987).

In some cases, experimental data have shown that there is a close relationship between pp Gpp levels in cells and antibiotic production. / Ochi, K., J. Gén. Microbiol. 132: 2621-2631 (1986); Ochi et al., J. Antibiot. 41: 1106-1115 (1988).

It is difficult to study and understand the regulation that the change in GTP level is related to the so-called. with a stringent response, that is, the phenomenon that microorganisms are known as "microorganisms" to reduce the nitrogen or carbon source of the medium. they respond by decreasing the synthesis of stable RNAs, in parallel with decreasing GTP levels. / Freese et al. Developmental Bioi. 102: 438-451 (1984). However, this answer is the so-called. rel in A and rel C mutant strains.

It is also known that the synthesis of ppGpp and pppGpp is reduced by the use of very low concentrations of chloramphenicol in strains of the microorganism that are otherwise sensitive. At this concentration (0.7-4 pg / ml), chloramphenicol does not inhibit growth yet. / Ochi, K and Freese, E. J. Gene. Microbiol 129, 3709-3720 (1983), Ochi, K .: J.Gen. Microbiol 132: 2621-2631 (1986).

When used in higher concentrations, chloramphenicol stops their growth by inhibiting protein synthesis.

In most cases, the development of chloramphenicol resistance is not accompanied by an increase in ppGpp and pppGpp in the mutant, but in a minority of cases. Similarly, an increase in ppGpp and pppGpp levels is not always associated with an increase in antibiotic biosynthesis / Ochi, K .. J. Bact. 169: 3608-3616 (1987). However, the lack of synthesis of the above nucleotides always results in a loss of antibiotic productivity.

Thiosteptin and thiopeptin antibiotics of very similar structure have been successfully used several times to isolate rel mutants. Experimental experience shows that thiostreptone and A significant proportion of thiopeptin resistant mutants are rel mutants in which, after depletion of the carbon source and / or nitrogen source of the medium, there is no reduction in GTP, virtually undetectable ppGpp and pppGpp, and loss of antibiotic productivity. The thiostrepton-producing strain Streptomyces laurentii, which is apparently resistant to self-produced thiostrepton, is equally obviously not mutant because it has the capacity for antibiotic production (and thus the synthesis of ppGpp). Thus, it is expected that increased levels of ppGpp and pppGpp in the strain will result in an increase in antibiotic productivity in some chloramphenicol resistant mutants.

In our experiments, the strain of Streptomyces laurentii producing thiostrepton was subjected to mutagenic treatment and chloramphenicol resistant mutants were isolated. Resistance of the parent strain to chloramphenicol is about 30 pg / ml, while that of the mutants is> 200 pg / ml. The mutagenic treatment is carried out in a manner known in the art by UV irradiation, but other mutagens well known in the art may be used, e.g. N-methyl-N-nitro-N-nitrosoguanidine, radioactive radiation, nitrogen mustard, etc.

UV irradiation was performed with a 30 W germicidal lamp by exposing 10 ⁹ spores from a distance of 20 cm to UV irradiation for 10, 20, 30 minutes, and then irradiating the spores with selective medium and culturing them. Generally, after 10 minutes of UV irradiation, the number of viable spores decreased from 10 to ⁹ ° C.

The mutagenized spores were cultured on a selective medium containing 200 pg / ml chloramphenicol, labeled G / 1 and composed as follows:

EN 209 929 Β glucose 10.0 g yeast extract 2.5 g dipotassium hydrogen phosphate 1.0 g agar 20.0 g distilled water per 1000 ml

The pH is adjusted to 7.0-7.2 before autoclaving

NaOH.

After mutagenization, colonies grown on selective medium containing 200 μg / ml chloramphenicol were plated first on G / 1 medium containing no chloramphenicol and then again on selective medium to check the stability of the resistant mutants. The ability of stable chloramphenicol resistant mutants to produce thiostrepton was then tested. Among the resistant mutants, we found that thiosstreptone productivity was significantly higher than that of the parent strain. They account for about 10%.

One of these mutants, which had the most favorable property for industrial fermentation, was deposited in the National Collection of Agricultural and Industrial Microorganisms, NCAIM 0 01149. and the parent strain under NCAIM 001150. during 1991, IX. 3 (deposit instrument attached).

No. NCAIM 001149. The advantage of the strain is that it rapidly multiplies in cheap raw materials used in industrial fermentation, so that a greater amount of cells and thus a greater amount of antibiotics can be produced in the fermentation broth in the same time than the parent strain. A further advantage of the strain is that its spore formation and thus the production of the antibiotic are not inhibited by easily utilizable sources of carbon and nitrogen, so that they can also be used as a raw material for fermentation, thereby accelerating cell proliferation.

In view of the foregoing, the present invention provides for the preparation of a thiostrepton antibiotic comprising the isolation of a chloramphenicol resistant mutant of the parent strain Streptomyces laurentii NCAIM 001150, preferably a strain of NCAIM 001149 that assimilates, such as corn, corn, wheat, potato starch and / or glucose and / or dextrin and / or sucrose liquefied with amylase as well as assimilable nitrogen sources such as soy flour and / or corn jam and / or protein hydrolyzate such as yeast, and / or cultured in culture medium containing feed yeast and / or ammonium sulfate and / or urea and mineral salts at 20-40 ° C, preferably 28 ° C, and isolating the thiostrepton from the fermentation broth in a known manner. In the process of the present invention, at the end of the fermentation, the antibiotic content of the fermentation broth is between 1600 and 2200 µg / ml.

This production value is significantly higher than the results so far known. The highest so far reported in the literature was 1000-1100 pg / ml in a 10 liter batch fermentation (Applied Microbiology and Biotechnology, 25, 520-531 (1987)).

The antibiotic content of the fermentation broth and the final product was determined by agar diffusion as described in USP XXII using Bacillus subtilis ATCC 6633 and / or Staphylococcus aureus 6538P as test organisms and refer to a commercially available thiostrepton reference standard.

The HPLC analysis of thiostrepton end product was also performed as follows:

Column: LiChrosfer RP 8 5 um (250mm x 4mm)

Eluent: 72% methanol + 28% water

Detection: 280 nm

Flow rate: 1.0 ml / min

Injected volume: 20 μΐ

The following examples illustrate fermentation with Streptomyces laurentii strain NCAIM 001149 without limiting the invention.

Comparative microbiological characterization of microorganism strains

The microbiological characterization of the parent Streptomyces laurentii strain and the mutant strain isolated after the mutagenic treatment were described by the International Streptomyces Project (ISP) and Shirling et al. Int. J. Syst. Bact., 16: 313340 (1966), according to the available materials and devices.

The results of the microbiological characterization of the parent and mutant strains are summarized below. Comparison of the data showed no significant difference between the two strains.

Starting strain Mutant strain 1. Colony morphological characteristics on different media 1.1. Yeast extract glucose agar medium sporulation, aerial mycelium white, pale pinkish white, backside yellowish brown, soluble pigment pink sporulation stronger, aerial mycelium pale pink, backside brown, soluble pigment pink 1.2. Oatmeal agar sporulation is good, aerial mycelium pale gray-pink, backside color not typical sporulation good, aerial mycelium yellowish pink, backside beige pink 1.3. Synthetic agar good sporulation, aerial mycelium pale beige pink, backside pink, no soluble pigment sporulation good, aerial mycelium pink, backside orange-pink 2. Physiological characteristics no melanin production on casein agar no melanin production on casein agar 3. Microscopic image (shake culture, 18 hours, 28 ° C, tryptone yeast extract in culture medium) grayish-pink soluble pigment, fragmented, mycelial arthrospores and longer to shorter hyphae soluble pigment darker mycelium fragmented, spores and short hyphae

HU 209 929 Β

Starting strain Mutant strain 4. Biochemical characteristics Compounds that can be used as the sole carbon source glucose xylose + + maybe actose (+) + melibóz + + sucrose (+) + lactose + + mannitol + + inositol - - arabinose - - sorbitol - - rhamnose - - fructose - - raffinose - -

EXAMPLES 25

Example 1

A frozen spore suspension of the strain Streptomyces laurentii NCAIM 001150 (-20 to -30 ° C) was thawed at room temperature and inoculated with 30 ml of the following inoculum.

The pH of the medium was adjusted to 7.4 with NaOH prior to autoclaving, and the pH to 6.3-6.5 after autoclaving (121 ° C for 20 minutes).

Composition of the trace element solution:

CoCl ₂

CuCl ₂

MgCl ₂

CaCl ₂

FeCl ₂

ZnCl ₂

MnCl ₂

NaCl

HCl (0.05%) distilled water

5g 0.027 g

35.5 g 0.59 g 0.48 g 0.20 g 0.30 g 0.25 g 8ml / L

Make up to 1000 ml

Add 50 ml of TF-17 medium to a 500 ml Erlenmeyer flask. The inoculated TF-17 medium was cultured at 28 ° C in a 180 / min shaker for 96 hours. At the end of the fermentation, the thiostrepton content of the fermentation broth is 700-1100 μg / ml.

Example 2

The frozen spore suspension of Streptomyces laurentii NCAIM 001149 (-20 to -30 ° C) was thawed at room temperature and inoculated with 0.5 ml of Ti-15 medium described in Example 1. In the following, all the procedures of Example 1 are followed. At the end of the laboratory fermentation, the thiostrepton content of the fermentation broth is 1500-1900 pg / ml.

Example 3

medium (designated Ti-15): Streptomyces laurentii strain NCAIM 0 01149 soybean meal 15g frozen spore suspension 3.5 ml soluble starch 15g Inoculate 800 ml of medium in 4-L flasks with 1 ml. glucose 50g 35 followed by preparation of the inoculum in Example 1 corn jam (50%) 10g we continue. The inoculum was inoculated with 1 m ³ of sterile calcium carbonate 1g Ti-16 in a 2 m ³ fermentor. cobalt chlorides (Π) 5 mg Composition of Ti-16 medium: polypropylene glycol 600 mg soybean meal 15 kg distilled water Make up to 1000 ml 40 wheat starch 15 kg Before sterilization, the pH is adjusted to 7.4 with NaOH, glucose 50 kg (121 ° C, 20 minutes) the pH of the medium is 6.3-6.5. corn jam (50%) 10 kg Add 50 ml of Ti-15 medium to 500 ml Er- calcium carbonate 2 kg in a lenmeyer flask. Preparation of the inoculum at 28 ° C cobalt chlorides (Π) 5g On a 180 / min spinner (stroke 2.5 45 poly-propylene glycol 0.6 kg cm), continued for 72 hours. The culture thus obtained 2 alpha-amylase (BAN 240) 100 ml ml of the following medium (signal tap water added to 10001 TF-17): The pH of the medium before sterilization is adjusted to 7.6-7.8, soybean meal 15g after sterilization (121 ° C for 30 min) the pH is 6.2-6.5. Your- wheat starch 15g 50 culture at 28 ° C with an internal overpressure of 0.7 bar, corn jam (50%) 30g We continue with aeration of 60 m ³ / hour, 72 hours. glucose 30g The inoculum thus prepared is inoculated at 30 m Fermen ³ yeast feed lg in a sterile broth volume of 20 m ³ , ammonium sulfate 4.3 g having the following composition: calcium carbonate 2g 55 soybean meal 300 kg potassium dihydrogen phosphate 52g wheat starch 300 kg alpha-amylase (BAN 240) 0.1 ml glucose 600 kg polypropylene glycol 600 ml yeast feed 20 kg trace element solution 1 ml ammonium sulfate 86 kg distilled water Make up to 1000 ml 60 corn jam (50%) 600 kg

EN 209 929 Β calcium carbonate 80 kg polypropylene glycol 13 kg potassium dihydrogen phosphate 1 kg alpha-amylase 2 liters magnesium chloride 710 g tap water 20000 liters

The medium was adjusted to pH 7.6-7.8 before sterilization, and after sterilization (121 ° C, 45 minutes), the pH was 6.2-6.5.

Fermentation was continued at 28 ° C for 4 days at an internal pressure of 0.5 bar with aeration of 1200 m 3 / h. The agitator speed is 50 rpm for the first 20 hours, then 100 rpm until the end of the fermentation. From the 20th hour onwards, the pH was adjusted to 6.2 to 6.8 by addition of sulfuric acid. At the end of the fermentation, the active substance content of the fermentation broth is 1500 μg / ml.

The thiostrepton is isolated from the fermentation broth as follows. At the end of the fermentation, cells are harvested from the fermentation broth cooled to 20 ° C with a separator. From 20 m ^{3 of} fermentation broth, 1500 kg of wet cell mass is obtained. The harvested cells were resuspended in 5000 L of methanol, and after centrifugation and washing with methanol, the methanol suspension and wash were repeated with 1500 L of methanol. The methanol cell weight collected by centrifugation was 700 kg and extracted with 2400 liters of chloroform: methanol = 1: 1. The extract was evaporated to one-tenth of the original volume (about 250 L) at 30-34 ° C in vacuo and the thiostreptone crystallized at 4 ° C by addition of an equal volume of methanol. The crystals were collected by filtration, dried in a vacuum oven at 25 ° C and then pulverized. Above 20 m ³ of fermentation broth obtained 13 kg of thiostrepton with a purity by HPLC of 98%, the biological activity of 1350 pg / ml.

Claims (4)

PATIENT INDIVIDUAL POINTS 1. A method for producing a thiostrepton antibiotic by microbial aerobic fermentation, a carbon and nitrogen source assimilable with a strain of Streptomyces laurentii, and a mineral salt containing cultures of chloramphenicol-resistant Streptomyces laurentii NCAIM 001149 at 20-35 ° C. the antibiotic is isolated from the cells. 2. The process according to claim 1, wherein the fermentation is preferably carried out at 28 ° C. 3. Azl. Method according to claim 1 or 2, characterized in that the strains of Streptomyces are cultivated on soybean meal, corn starch, glucose, alpha-amylase-distilled wheat starch, ammonium sulfate, calcium carbonate, feed yeast. Process according to any one of claims 1 to 3, characterized in that the thiostrepton antibiotic is extracted from the cells collected from the fermentation broth with a separator of chloroform: methanol = 1: 1, and crystallized by the addition of methanol after evaporation of the extract.