The present invention relates to an improved fermentation process for preparation of ascomycin by controlling the physico-chemical parameters.
Ascomycin or FK-520 of Formula I, is a well-known antifungal antibiotic, which inhibits filamentous fungi, e.g. Penicillium chrysogenum and is also active against acid-fast bacteria. Ascomycin also finds use as a starting material for the synthesis of pimecrolimus, which is used for the treatment of atopic eczema.
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Ascomycin (FK-520) is usually produced by microbial fermentation whereby there is formation of homologous compound FK-523 from which ascomycin has to be separated and purified. The presence of FK-523 in ascomycin is undesirable as it has lesser immunosuppressive activity than ascomycin. FK-523 has methyl group at C-21 resulting from the substitution of a propionate for a butyrate during polyketide synthesis. The separation and purification of ascomycin from FK-523 produced by fermentation methods is often complicated as they have almost the same number of carbon atoms and are similar to each other in physical properties such as solubility and affinity to solvents.
US Patent No 3,244,592 discloses the isolation of ascomycin from Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891, MA 6475) and its use as an antifungal antibiotic.
US Patent No 4,894,366 discloses a method for production of ascomycin and FK-523 by fermentation using Streptomyces subspecies yakushimaensis No. 7238 (FERM BP-928) wherein ascomycin is isolated and purified from the fermentation broth by conventional methods.
Hatanaka et al. J. Antibiotics 41., 1592-1601, (1988) disclose a method for production of ascomycin (FK-520) as an immunosuppressant from Streptomyces subspecies yakushimaensis No. 7238 (FERM BP-928, MA6531) and purification thereof by extraction from the fermentation broth followed by crystallization.
Junker et al. Biotechnology and Bioengineering 59, 595-604, (1998) disclose a method for production of ascomycin from Streptomyces hygroscopicus by valine feeding during fermentation. Ascomycin produced under valine-fed conditions is reportedly contaminated with about 3.5% of the FK-523 homolog.
Junker et al. Biotechnology and Bioengineering 60, 580-588, (1998) disclose a method for production of ascomycin using a valine-overproducing Streptomyces hygroscopicus mutant (MA7040) using soyabean oil and ammonium sulphate to optimize secondary metabolite formation.
The prior art methods basically target two approaches to control the level of FK-523 homolog impurity in ascomycin produced by fermentation technique. First approach is the separation of FK-523 impurity from ascomycin by selective extraction from the fermentation broth and purification by crystallization. Second approach is by valine feeding during fermentation or employing a valine-overproducing Streptomyces strain. FK-523 levels of 2-12% are observed in ascomycin depending upon the quantity of valine fed.
The additional recovery methods when applied to purify ascomycin do not lead to selective removal of the FK-523 impurity and there occurs significant loss of ascomycin due to close structural similarity between ascomycin and FK-523. Valine feeding leads to significant reduction in the yield of ascomycin to the tune of about 20%.
The present inventors after extensive studies have surprisingly found that ascomycin having significantly reduced levels of FK-523 homolog impurity can be produced by controlling the physico-chemical paramteres such as incubation temperature and agitation rates during fermentation. The present process is cost effective, commercially scalable without compromising on the yield of ascomycin. The present process offers a simpler means of controlling the FK-520 impurity during the fermentation stage itself and is a marked improvement over the prior art methods for production of ascomycin by fermentation.
The present process uses ascomycin hyper producer strain of Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891). It is to be understood that the production of ascomycin by the present process is not limited to the use of the particular microorganism described herein, which is given for illustrative purpose only. The present invention also includes the use of any mutants, which are capable of producing ascomycin including natural mutants as well as artificial mutants, which can be produced from ascomycin producing microorganism or mutant thereof by conventional methods.
Table 1 represents a comparison between the prior art methods for production of ascomycin and the present invention with respect to the fermentation conditions and level of FK-523 contamination in ascomycin. The present inventors have surprisingly observed that FK-523 formation during fermentative production of ascomycin can be reduced from about 20% to 4% at an incubation temperature of about 24-32°C and an agitation rate of about 2.0-4.0 m/s.
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A first aspect of the present invention provides a fermentation process for producing ascomycin, wherein the said process comprises of,
(a) incubating ascomycin producing microorganism in a nutrient medium at temperature of 24-32°C and agitation rate of 2.0-4.0 m/s,
An ascomycin producing Streptomyces strain was incubated in a nutrient medium for about 180-240 hours at 24-32°C at an agitation rate of 2.0-4.0 m/s. The level of ascomycin and FK-523 homolog impurity in fermentation broth was determined using a slight modification of the HPLC method reported in Biotechnology and Bioengineering 59: 595-604, 1998. Ascomycin can be isolated from the fermentation mixture either using the whole broth or using the mycelium and the filtrate obtained therefrom. More preferably the incubation temperature was maintained at about 26-30°C at an agitation rate of 2.0-2.8 m/s. Most preferably the incubation temperature was maintained at about 28°C at an agitation rate of 2.4 m/s.
EXAMPLE 1
Streptomyces hygroscopicus subspecies ascomyceticus mutant (ATCC 14891) was grown and maintained on yeast extract malt extract agar slants. The surface of the slant was scraped and used to inoculate a 250 ml shake flask containing 35 ml of a sterilized seed medium containing dextrose (1.0 g/L), dextrin (10 g/L)), milk (2 g/L), cotton seed meal (2.5 g/L), casein enzyme hydrolysate (5 g/L), MgSO4.7H2O (0.05 g/L), FeSO4.7H2O (0.025 g/L), NaCI (0.5 g/L), CaCI2 (0.02 g/L), ZnSO4.7H2O (0.01 g/L), MnSO4.7H2O (0.005 g/L) and phosphate buffer (2ml /L; pH 7.0). The pH of the seed medium was adjusted to 7.2. The flask was incubated for 48 hours at 28°C on a rotary shaker. An aliquot of the resulting seed culture was used to inoculate a shake flask containing presterilized production medium consisting of dextrin (140 g/L), cotton seed meal (14 g/L), soya bean meal (10 g/L), glycerol (10 g/L), soya peptone (10 g/L), polyethylene glycol (12.5 g/L), potassium phosphate (0.8 g/L), calcium carbonate (1.5 g/L) and the pH was adjusted to 7.2. The flask was incubated at 24°C on a rotary shaker. Ascomycin: 350-400 mg/L (by HPLC) FK 523 content: 18 % (by HPLC).
EXAMPLE 2
The strain propagation and inoculum preparation methods were same as described in
Example 1. 10 ml of inoculum obtained in Example 1 was added to 10 L bioreactor
containing seed medium described in Example 1, except that the milk powder and

cottonseed meal concentration was 3 g/L each and additionally calcium carbonate (0.5 g/L) was added. The medium was sterilized at 121°C for 30 minutes. After 47 hours of incubation, seed culture was used to inoculate 20 L production bioreactor containing dextrin (30 g/L), cottonseed meal (10 g/L), soya bean meal (10 g/L), glycerol (10 g/L), soya peptone (10 g/L), polyethylene glycol (12.5 g/L), potassium phosphate (0.8 g/L) and calcium carbonate (1.5 g/L). A dose of dextrin solution was fed during fermentation when pH increased beyond 7.6-7.8. Fermentation was run at 24°C. The agitation rate was increased to 3.2-4.0 m/s during growth phase. The agitation rate was decreased during the production stage to 2.4-2.8 m/s. Ascomycin: 350-400 mg/L (by HPLC) FK 523 content: 17.8 % (by HPLC).
EXAMPLE 3
The strain propagation, inoculum preparation method and initial fermentation batch conditions were same as described in Example 1 and 2 except that the agitation rates during growth and production phase were 2.7-3.3 m/s and 2.0-2.4 m/s respectively. Ascomycin: 350-400 mg/L (by HPLC) FK 523 content: 9.29 % (by HPLC).
EXAMPLE 4
The procedure of Example 1 was followed for microorganism propagation and fermentation except that the production shake flasks were incubated at 28°C. Ascomycin: 350-400 mg/L (by HPLC) FK523 content: 7%.
EXAMPLE 5
The procedure of Example 1 was followed for microorganism propagation and fermentation except that the production shake flasks were incubated at 30°C. Ascomycin: 350-400 mg/L (by HPLC) FK523 content: 4.8%.
EXAMPLE 6
The procedure of Example 1 was followed for microorganism propagation and fermentation except that the production shake flasks were incubated at 32°C. Ascomycin: 350-400 mg/L (by HPLC) FK523 content: 5.0%.
EXAMPLE 7 The strain propagation and inoculum preparation methods were same as described in Example 1. 100 ml of inoculum obtained in Example 1 was added to 100 L seed fermenter containing seed medium comprising components as described in Example 2. After 48 hours of incubation, 40 L of seed was used to inoculate 400 L production medium comprising components as described in Example 2. The fermentation was run at 28°C. A dose of dextrin solution was fed during fermentation when pH increased beyond 7.6-7.8. Agitation rate during growth and production phase was 2.7-3.3 m/s and 2.0-2.4 m/s respectively.
Ascomycin: 350-400 mg/L (by HPLC) FK 523 content: 4.7 % (by HPLC).
While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

WE CLAIM :
1. A fermentation process for producing ascomycin, wherein the said process comprises
of,
(a) incubating ascomycin producing microorganism in a nutrient medium at a temperature of 24-32°C and agitation rate of 2.0-4.0 m/s.
2. A process according to claim 1 wherein the incubtation temperature is 26-30°C.
3. A process according to claim 2 wherein the incubtation temperature is 28°C.
4. A process according to claim 1 wherein the agitation rate is 2.0-2.8 m/s.
5. A process according to claim 4 wherein the agitation rate is 2.4 m/s.
6. A process according to claim 1 wherein the microorganism is an ascomycin hyperproducer or mutant thereof.
7. A process according to claim 6 wherein the microorganism is Streptomyces species or mutant thereof.
8. A process according to claim 7 wherein the microorganism is Streptomyces hygroscopicus subspecies ascomyceticus (ATCC 14891).
9. A process according to claim 1 wherein the nutrient medium comprises soya bean meal, dextrose, dextrin, milk, cotton seed meal, casein enzyme hydrolysate, CaCl2 and CaC03.
10. A fermentation process for producing ascomycin as described herein and exemplified in the examples.