Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Patent
1997-04-18
1999-01-05
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
435 713, 435 42, 435171, 4351722, 4352541, 435911, 530317, 530321, C12P 2104
Patent
active
058561412
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a microbial process for making a highly productive fusant of Tolypocladium inflatum, a producer strain of cyclosporin A with immunosuppressive property and more particularly, to a method of producing cyclosporin A by submersed fermentation method comprising the steps of; Based upon a wild strain Tolypocladium inflatum which produced a small amount of cyclosporin A, developing amino acid-dependent mutant strain induced by UV radiation; conjugating other two amino acid-dependent mutants; making a highly productive fusant strain of cyclosporin A in parallel with increasing demanding of amino acid and organic nitrogen-source; and establishing the suitable condition and method of culture to these fusant to obtain the cyclosporin A by submersed fermentation method.
DESCRIPTION OF THE PRIOR ART
In more detail, Tolypocladium inflatum Gams NRRL 8044, a producer strain of cyclosporin A, is fungus and cyclosporin A which this strain produces is cyclic peptide consisting of 11 amino acids and molecular weight is 1,201, molecular formula is C.sub.62 H.sub.111 N.sub.11 O.sub.12, and there are HELVETICA ACTA, 70,13(1987)! 3R,4R)-3-hydroxy-4-methyl-2-(methyl-amino)-6-octenoyl}-L-2-aminobutyryl-N- methyl-glycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-O-ala nyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl!, is known to have antifungal, antiparasitic and antiinflammatory properties as well as a potent immunosuppressive property, and is important in the treatment of Allergy 38, 9 (1986)!.
In general, the productive capacity of a producing strain is very important in producing secondary metabolites by fermentation of microorganisms.
Sesquicillopsis rosariensis G. ARNOLD F605 with 3150 mg/L and Tolypocladium inflatum Wb6-5 with 1100 mg/L(U.S. Pat. No. 5,256,547, 1993) are known as the highest productive strains among known cyclosporin A-producing strains.
The highly productive Cyclosporin A-forming strain among known strains has not been reported since cyclosporin A was for the first time isolated by that the highly productive mutant of Tolypocladium inflatum has been developed actively and used as industrial producing strain.
SUMMARY OF THE INVENTION
The inventors made a highly productive cyclosporin A-forming mutant and invented a method for its fermentation. This mutant is characterized by producing cyclosporin A in high concentration, thus requiring large amounts of L-valine and L-leucine as well as organic nitogen source to produce cyclosporin A.
Cyclosporin A is cyclic peptide consisting of 11 amino acids; valine at position of 5, 11, leucine at position of 4, 6, 9, 10 and their derivatives. Tolypocladium inflatum NRRL 8044, a cyclosporin A-producing strain, has known to produce cyclosporines selectively by adding special amino acids, the structural constituents of cyclosporines to a culture (1982)!.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
FIG. 1A, 1B, and 1C are chromatograms of cyclosporin A, pure product and cyclosporin A produced by wild strain KD01 and the high-producing fusant KD461 of Tolypocladium inflatum, respecti vely.
FIG. 2 is a photographs showing a colonial morphology of wild strain KD01 and the fusant KD461 of Tolypocladium inflatum cultured in a malt-yeast extract medium, respectively.
FIG. 3A and 3B are photographs (A) and (B) showing a morphology of hyphae, culturing wild strain KD01 and the fusant KD461 of Tolypocladium inflatum in a liquid culture medium for 192 hours, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The inventors made the targeted fusant with amplified cyclosporin A-producing capability, where it comprises; as a method of amplifying production of cyclosporin A, mutating a poor producing strain of cyclosporin A, wild strain of Tolypocladium inflatum isolated from soil making L-valine-dep
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Choi Byoung Tack
Kim Jung Woo
Lee Jin Man
Lee Kwang Moo
Min Kyeong Bok
Chong Kun Dang Corp.
Lilling Herbert J.
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