CLIAMS:We Claim,

1) A mutant strain of Glarea lozoyensis ATCC 74030, which has accession No JCCC-1097, wherein said mutant strain produces between 2.1 to 2.24gm/l Pneumocandin B0
2) The mutant strain according to claim 1, wherein the Pneumocandin C0 level is less than 2.0%.
3) The mutant strain according to claim 1, wherein said strain is mutated by treating said Glarea lozoyensis ATCC 74030 with mutagens.
4) The mutant strain according to Claim 2, wherein said mutant stored in glycerol stock at -800C.
5) The mutant strain according to Claim 1, wherein said mutant strain is cultivated in suitable fermentation medium.
6) The mutant strain according to claim 4, wherein said fermentation pH in a range of 5.2-5.4.
7) The mutant strain according to claim 4, wherein said fermentation temperature in a range of 22-280c
8) The mutant strain according to claim 6, wherein said fermentation suitable temperature is 24 0c
9) The mutant strain according to claim 4, wherein said mutant cultivated in a Sporulation media.
10) The mutant strain according to claim 8, wherein said sporalution media is potato dextrose agar.
11) The mutant strain according to claim 4, wherein said spore suspension is inoculated in Seed Medium.
12) The mutant strain according to claim 10, where is said seed medium composed of Mannitol, Yeast extract, tryptone, soya flour, KH2PO4, and (NH4)2SO4.
13) The mutant strain according to claim 4, wherein said seed culture is inoculated in production medium.
14) The mutant strain according to claim 12, wherein said production medium composed of mannitol, yeast extract, tryptone, L-Proline, soya 0il, KH2PO4, and (NH4)2S04.
15) The mutant strain according to claim 4, wherein the fermentation is done in fed batch mode and feeding started from 144 hrs of fermentation to reduce the impurity, Pneumocandin C0.
16) The mutant strain according to claim 14, wherein said feed is mannitol, ammonium sulphate and L-proline.
17) The mutant strain according to claim 1, wherein said mutant strain produces 2.1 to2.24 gm/l of Pneumocandin B0.
,TagSPECI:Technical Field:
The patent relates to isolation of a mutant strain of Glarea lozoyensis and biotechnology preparation of Pneumocandin B0 with improved process, in concentration of 2.1 to 2.24 gm/l by aerobic fermentation with cultivation time of approximately 300 hrs at temperature of 24oC with reduced level of structural analogues especially Pneumocandin C0.

Background Art:

Pneumocandin, a type of echinocandin derived from the fermentation of the fungus, have been successfully used to develop an antifungal drug. Pneumocandin which is similar to the echinocandin and aculeacin class of natural products in that the amino acids that compose the polar hexapeptide nucleus each possess one or more hydroxyl groups. Similarly, the Pneumocandin also possess a non-polar, amide-linked acyl side chain. Pneumocandin B0 is produced as a secondary metabolite by fermentation of the fungus Glarea lozoyensis (U.S. Pat. Nos. 5,194,377 and 5,202,309) which is precursor for Caspofungin Acetate (CANCIDAS), a semi synthetic lipopeptide Echinocandin B derivative currently being sold in US as an antifungal agent for intravenous administration.

Pneumocandin B0 molecular weight 1065 Dalton is an active or key intermediate for antifungal drug Caspofungin Acetate. Caspofungin Acetate is a complex, semi synthetic molecule with lipid and peptide substructures. It is derived from a fermentation product (Pneumocandin B0) of the anamorphic fungus Glarea lozoyensis. It inhibits the synthesis of an essential component of the cell wall of many fungi, the glucose homopolymer ß-(1, 3)-D-glucan, which is absent in mammals. This interferes with fungal cell wall synthesis, leading to instability and death of the fungal cell.

The first member of the Pneumocandin class to be isolated was Pneumocandin A0, a compound that is less hemolytic than other members of the naturally occurring echinocandin. Pneumocandin B0 appears to be the most potent glucan synthase inhibitor: However, its in vitro and in vivo antifungal activity is comparable to that of Pneumocandin A0. Pneumocandin B0 differs from Pneumocandin A0 only by absence of a methyl on one of the proline rings. (Current Pharmaceutical Design, 1996, vol.2, No, 2. Turner and Rodriguez). The major Pneumocandin produced by wild-type Z. arboricola is Pneumocandin A0, whereas Pneumocandin B0 is a minor component of the fermentation (Schwartz et al., 1992. J. Antibiotics 45:1853-1866). Pneumocandin B0 and Pneumocandin C0 are isomers which differ by the position of one hydroxyl group at a proline residue only


Structural Analogues of Pneumocandin B0

R1 R2 R3 R4 R5 R6 R7
1 Pneumocandin A0 CH3 OH OH OH CH3 OH OH
2 Pneumocandin A1 CH3 OH OH OH CH3 H OH
3 Pneumocandin A2 CH3 OH H H CH3 OH OH
4 Pneumocandin A3 CH3 OH OH H CH3 H H
5 Pneumocandin A4 CH3 OH H H CH3 H H
6 Pneumocandin B0 H OH OH OH CH3 OH OH
7 Pneumocandin B1 H OH OH OH CH3 H OH
8 Pneumocandin B2 H OH H H CH3 OH OH
9 Pneumocandin B5 H OH OH H CH3 OH OH
10 Pneumocandin B6 H OH H OH CH3 OH OH
11 Pneumocandin C0 OH H OH OH CH3 OH OH
12 Pneumocandin D0 OH OH OH OH CH3 OH OH
13 Pneumocandin D2 OH OH H H CH3 OH OH
14 Pneumocandin E0 H H OH OH CH3 OH OH
15 B0 Serine Analogue H OH OH OH CH3 H OH
16 B5 Serine Analogue H OH OH H H OH OH

During the fermentation of Pneumocandin B0, proline supplementation resulted in a dose-dependent increase in the levels of Pneumocandin B0 and E0 where as Pneumocandin C0 and D0 decreased as a function of proline level. However, the C0 analogue was more than 2.6% in all the cases and the actual titre value of Pneumocandin B0 was never disclosed (Journal of Industrial Microbiology & Biotechnology, 2001, 26, 216-221)

A high total Pneumocandin titre (B0 + C0) with a low percentage of the structural isomer Pneumocandin C0 was said to have been achieved with high residual fructose concentration. Midcycle addition of NaCl and Na2SO4 at low residual fructose concentration also is reported to have resulted in reduction in Pneumocandin C0 levels. However the C0 analogue was more than 4.2%. (Applied Microbial Biotechnology, 2000, 54, 814-818)

US8431383 discloses mutagenized strain of Glarea Lozoyensis and which is high yielding producing little impurities in fermentation. However, impurity levels generated in fermentation were never disclosed.

W02013/104576 discloses that improvement in biomass growth, productivity and decrease in unwanted impurities can be obtained with increased trace metal ion concentration. However neither the titre value nor the level of reduction in impurities was disclosed.

Disclosure of the Invention:

The patent is related to biotechnology preparation of Pneumocandin B0, the novel microorganism is a mutant strain of Glarea lozoyensis ATCC 74030. The mutant strain is deposited at MCC, Pune, under the accession no of JCCC-1097. The present invention is focused on increasing the Pneumocandin B0 titer and reducing the level of structural analogues especially Pneumocandin C0 by strain improvement and process optimization. The mutant strain Glarea lozoyensis produces Pneumocandin B0 in concentration of 2.1 to 2.24 gm/l in fermentation broth till 300 hours of cultivation in production media at 240C. The fermentation broth is worked up as per the patent 2659/CHE/2013, the Pneumocandin C0 levels were found to be below 2.0% in the product.

Glarea lozoyensis JCCC-1097, can be obtained by applying a classical mutation-inducing technique on the parent strain, Glarea lozoyensis ATCC 74030. For example, irradiation of the microorganisms with ultraviolet light or treatment with mutagen, such as N-methyl-N-nitro-N-nitrosoguanidine, ethyl methane sulfonate and methyl methane sulfonate are suitable methods of inducing mutation. The desired mutant, Glarea lozoyensis JCCC-1097, is obtained as a strain capable of producing Pneumocandin B0 at the highest concentration level among all mutant strains.

In this embodiment, the microorganisms are cultivated by the following process for producing Pneumocandin B0 at the highest concentration level, the process comprising the steps of:
1) Cultivating the microorganism in a spore medium
2) Eluting the spore and preserving the spore suspension in glycerol stock
3) Cultivating the spores suspension in seed medium
4) Cultivating the seed culture in the fermentation medium to produce Pneumocandin B0
5) Feed addition from 144 hour to control impurities and to increase product concentration.
The spore medium can be potato-Dextrose agar (PDA) Medium. The PDA medium contains of potato extract, Dextrose, and agar, preferably, the PDA medium contains 20% of potato extract, 2% of Dextrose, and 2% of agar.

The seed medium contains mannitol, yeast extract, tryptone, soya flour, potassium dihydrogen phosphate, and ammonium sulphate. Preferably, the seed medium contains 2% of mannitol, 0.2% yeast extract, 0.2% tryptone, 1% soya flour, 0.75% of potassium dihydrogen phosphate, 0.75% of ammonium sulphate and pH about 5.2-5.4.

The fermentation medium contains mannitol, yeast extract, tryptone, potassium dihydrogen phosphate, and ammonium sulphate, trisodium citrate, and L-proline. Preferably, the fermentation medium contains 10% of mannitol, 0.8% yeast extract, 2% tryptone, 1% of potassium dihydrogen phosphate, 1% Soya Oil, 0.5% of ammonium sulphate, 0.3% of trisodium citrate, 0.8% of proline and pH about 5.2-5.4.

During fermentation proline, mannitol and ammonium sulphate feed is added during the production phase of Pneumocandin.

The fermentation broth was analyzed on HPLC (Agilent 1100 Series). The normal phase analysis employed a silica column (100 Å pore diameter X 5 µm particle diameter; 4.6 mm id X 250 mm). Elution was isocratic with a mobile phase composed of 84/9/7 (EtOAc/MeOH/water) at 1.5 mL/min, and the effluent was monitored at 278 nm.

EXAMPLE-1:
The Culture of Glarea lozoyensis ATCC 74030 was grown for sporalution on Potato dextrose agar (PDA) at 240C for 8-12 days. The spore suspension was transferred to phosphate buffer and spores were collected by centrifuge. The collected spore’s suspension was mutagenized physically. e.g. irradiating with UV rays at a distance of 15 to 45 cm for different time intervals or chemically e.g. by treating with N-methyl-N-nitro-N-nitrosoguanidine (NTG), ethyl methane sulfonate and methyl methane sulfonate at a different concentration of 10 µg to 1000 µg/ml for different time intervals. The mutated spores were cultivated on PDA at 240C for 8-12 days. A total of 123 mutants were selected and cultivated to determine the productivity of Pneumocandin B0 titer value.

Table -1
Pneumocandin B0 Titer 0-0.5 gm/l 0.5-0.75 gm/l 0.75-1.0 gm/l 1.0-1.25 gm/l 1.25.0-1.5 gm/l

No Of mutated Glarea Lozoyensis ATCC 74030 64 28 16 11 7

64 strains gave a Pneumocandin titer of below 500 mg/l and 28 strains gave a Pneumocandin titer of 500 to 750 mg/l and 7 strains gave a Pneumocandin titer of 1250 to 1500 mg/l, as indicated in the above table.

One strain from 7 mutant strain having Pneumocandin titer 1250 to 1500 mg/l was found stable and produce Pneumocandin B0 at high concentration level. The mutant strain is deposited at Microbial Culture Collection (MCC) under accession number JCCC-1097 under the provisions of the Budapest Treaty for the International Recognition of the deposit of the microorganism for the purpose of patent Procedure.

Seed Preparation:
All mutant strain is cultivated by the same process for producing Pneumocandin B0. The spore suspension of Glarea lozoyensis JCCC-1097 was inoculated in the seed medium and incubated on shaker at 200rpm at the temperature of 22-280C for 48 to 72 hours. The Seed medium contains Mannitol, yeast extract, tryptone, Potassium dihydrogen phosphate, and ammonium Sulphate. Preferably, the seed medium contains 2% of Mannitol, 0.2% yeast extract, 0.2% tryptone, 1% soya flour, 0.75% of potassium dihydrogen phosphate, 0.75% of Ammonium Sulphate, and pH about 5.2-5.4.

Fermentation Process:
The seed culture of Glarea lozoyensis JCCC-1097 was inoculated in the production medium at 5 to 10% Inoculum size and shaken at 220 rpm at the temperature of 22-280C for 240 to 300 hours. The fermentation medium contains Mannitol, yeast extract, tryptone, potassium dihydrogen phosphate, ammonium Sulphate, trisodium citrate, soya oil and L-proline. Preferably, the fermentation medium contains 10% of Mannitol, 0.8% yeast extract, 2% tryptone, 1% of potassium dihydrogen phosphate, 1% soya Oil, 0.5% of ammonium sulphate, 0.3% of trisodium citrate, 0.8% of proline and pH about 5.2-5.4. Then the fermented broth was analyzed in HPLC to determine the Pneumocandin B0 concentration.

EXAMPLE-2:

Spore Preparation:
The spore suspension is inoculated on potato-dextrose agar (PDA) and incubated at 240C for 8-12 days. The PDA medium consist of potato extract, Dextrose, and agar, preferably, the PDA medium contains 20% of potato extract, 2% of Dextrose, and 2% of agar

Seed Preparation:
Then a loop of spores of the strain Glarea lozoyensis is inoculated in the seed media contains mannitol, yeast extract, tryptone, soya flour, potassium dihydrogen phosphate, and ammonium sulphate. Preferably, the seed medium contains 2% of mannitol, 0.2% yeast extract, 0.2% tryptone, 1% soya flour, 0.75% of potassium dihydrogen phosphate, 0.75% of ammonium sulphate and pH about 5.2-.5.4 and incubated on shaker at 200 rpm at the temperature of 22-280C for 48 to 72 hours.

Fermentation Process:
Prepared vegetative Inoculum in Erlenmeyer flask is seeded from 5 to 10% in to production step in 10liter fermentor with 7liter production volume with production media contains mannitol, yeast extract, tryptone, potassium dihydrogen phosphate, and ammonium sulphate, trisodium citrate, and L-proline. preferably, the fermentation medium contains 10% of mannitol, 0.8% yeast extract, 2% tryptone, 1% of potassium dihydrogen phosphate, 1% Soya Oil, 0.5% of ammonium sulphate, 0.3% of trisodium citrate, and 0.8% of L-Proline and. Temperature of fermentation was set to 240C, stirrer speed was set to 400 RPM and aeration rate was 0.5VVM. pH controlled and maintained between 5.25 to 5.4. Minimal concentration of dissolved oxygen was set to 20%. The dissolved oxygen level is first controlled by increasing the agitator rpm followed by air flow.

Additions of L-Proline start from 144 hrs of the culture in 2.5 gm/l per day. The Pneumocandin B0 was determined by HPLC. Concentration of Pneumocandin B0 in the fermentation broth was 1.78 gm/l. After workup, the Pneumocandin C0 level was found to be below 2.0%.

EXAMPLE-3:

Seed Preparation:
The spore suspension in glycerol stock is inoculated in the seed medium contains mannitol, yeast extract, tryptone, soya flour, potassium dihydrogen phosphate, and ammonium sulphate. preferably, the seed medium contains 2% of mannitol, 0.2% yeast extract, 0.2% tryptone, 1% soya flour, 0.5% of potassium dihydrogen phosphate, 0.5% of ammonium sulphate and pH about 5.2-5.4 and incubated on shaker at 200rpm at the temperature of 22-280c for 48 to 72hours.

Fermentation Process:
Prepared vegetative Inoculum in Erlenmeyer flask is seeded from 5 to 10% in to production step in 10liter fermentor with 7liter production volume with production media contains mannitol, yeast extract, tryptone, potassium dihydrogen phosphate, and ammonium sulphate, trisodium citrate, and L-proline. preferably, the fermentation medium contains 7.5% of mannitol, 0.8% yeast extract, 2% tryptone, 1% of potassium dihydrogen phosphate, 1% Soya Oil, 0.5% of ammonium sulphate, 0.3% of trisodium citrate, and 0.8% of proline. Temperature of fermentation was set to 240C, stirrer speed was set to 400 RPM and aeration rate was 0.5VVM. pH was controlled and maintained between 5.25 to 5.4. Minimal concentration of dissolved oxygen was set to 20%. The dissolved oxygen level is first controlled by increasing the agitator rpm followed by air flow.

Addition of 15gm/l mannitol, 0.1% ammonium sulphate and 0.2% L-proline was fed from 144hrs of the culture in batch mode for every 24 hours. The Pneumocandin B0 was determined by HPLC. Concentration of Pneumocandin B0 in the fermentation broth was 2.24gm/l with low level of impurities. After workup, Pneumocandin C0 level was found to be below 2.0%.

EXAMPLE-4:
The spore stability of Glarea lozoyensis JCCC-1097 on the Pneumocandin B0 was studied. The spore suspension in glycerol stock stored at -800C was thawed at 0day, 90days, 180days, 270days and 360days respectively , then the spores were cultivated in the same process as Example-3 and the Pneumocandin B0 were determined by HPLC. The spores of Glarea lozoyensis JCCC-1097 were found to have good stability. The results indicate the following table.2.

Table-2
Storing Days 0 90 180 270 360

Pneumocandin B0 titer gm/l 2.21 2.17 2.24 2.23 2.20