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United States Patent (19) 11 Patent Number: 5,856,141 Kim Et Al

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United States Patent (19) 11 Patent Number: 5,856,141 Kim Et Al USOO5856141A United States Patent (19) 11 Patent Number: 5,856,141 Kim et al. (45) Date of Patent: Jan. 5, 1999 54 PROCESS FOR MANUFACTURING OTHER PUBLICATIONS CYCLOSPORN A BY HIGHLY Traber et al., “Neu Cyclosporine aus Tolypocladium infla PRODUCTIVE FUSANT STRAIN tum) Die Cyclosporin K-Z”, Helyetica Chimica Acta 75 Inventors: Jung Woo Kim, Seoul, Kwang Moo 70:13–35 (1987). Lee, Anyang; Byoung Tack Choi; Jin Borel, “Editorial: Ciclosporin and Its Future', Prog. Allergy Man Lee, both of Seoul; Nak Kyu vol. 38:9-18 (1986). Sung, Euwang; Kyeong Bok Min, Riegger et al., “CycloSporin A, ein Immunosuppresive Seoul, all of Rep. of Korea Wirksamer Peptidmetabolit aus Trichoderma Polysporum (Link ex Pers.)", Helvetica Chimicia Acta 59(4): 1075–1092 73 Assignee: Chong Kun Dang Corp., Seoul, Rep. (1976). of Korea Kobel et al., “Directed Biosynthesis of Cylosporins”, Euro pean J. Appl Microtechnol., 14:237-240 (1982). 21 Appl. No.: 817,392 Anné et al., “Induced Fusion of Fungal Protoplasts Follow ing Treatment with Polyethylene Glycol”, J. Gen. Microbiol. 22 PCT Filed: Oct. 16, 1995 92:413-417 (1976). 86 PCT No.: PCT/KR95/00131 Peberdy et al., “Regeneration of Aspergillus nidulans Pro toplasts”, J. Gen. Microbiol. 69:325–330 (1971). S371 Date: Apr. 18, 1997 Primary Examiner Herbert J. Lilling S 102(e) Date: Apr. 18, 1997 Attorney, Agent, or Firm-Jacobson, Price, Holman & Stern PLLC 87 PCT Pub. No.: WO96/12032 57 ABSTRACT PCT Pub. Date: Apr. 25, 1996 The present invention relates to a proceSS for making a 30 Foreign Application Priority Data highly productive fusant of Tolypocladium inflatum, a pro ducing Strain of cycloSporin A with immunosuppressive Oct. 18, 1994 KR Rep. of Korea .................. 26689/1994 properties wherein the selection of the fusant KD461, (51) Int. Cl. ................................................ C12P 21/04 designed to produce a large amount of cycloSporin A, was 52 U.S. Cl. .......................... 435/71.1; 435/71.3; 435/42; made available by the following Steps of developing amino 435/171; 435/1722; 435/254.1; 435/911; acid-dependent mutants of Tolypocladium inflatum, wild 530/317; 530/321 strain isolated from soil, which mutants are induced by UV 58 Field of Search .................................. 435/71.1, 71.3, radiation; conjugating L-valine-dependent and L-leucine 435/911, 254.1, 171, 172.2, 42; 530/317, dependent mutants to promote the demand and utility of 321 L-Valine and L-leucine, precursors of cyclosporin A, together with an organic nitrogen-Source. The fusant KD461 56) References Cited produced has the following characteristics in comparison with wild strain: 1) it is slow growing in a Solid malt-yeast U.S. PATENT DOCUMENTS extract agar medium, b) the Surface of the colony is light grey in color and is therefore darker than the mild Strain, c) 4,943,529 7/1990 Van Den Berg et al. ........... 435/172.3 5,156,960 10/1992 Bokany et al. .............. ... 435/71.1 the backside of the colony is dark brown, and d) in a liquid 5,244,790 9/1993 Kim et al. ................................. 435/42 medium it has short and thick hyphae with many 5,256,547 10/1993 Rudat et al. .. 435/254.1 arthroSpore. 5,318,901 6/1994 Patchett et al. .. ... 435/71.1 5,639,852 6/1997 Rich et al. .............................. 530/321 5 Claims, 2 Drawing Sheets U.S. Patent Jan. 5, 1999 Sheet 1 of 2 5,856,141 U.S. Patent Jan. 5, 1999 Sheet 2 of 2 5,856,141 G. 3A 5,856,141 1 2 PROCESS FOR MANUFACTURING 8044, a cycloSporin A-producing Strain, has known to pro CYCLOSPORN A BY HIGHLY duce cycloSporines Selectively by adding special amino PRODUCTIVE FUSANT STRAIN acids, the Structural constituents of cycloSporines to a cul ture medium H. Kobel, European J. Appl. Microbiol Bio FIELD OF THE INVENTION technol 14:237-240, (1982). The present invention relates to a microbial process for BRIEF DESCRIPTION OF THE DRAWINGS making a highly productive fusant of Tolypocladium inflatum, a producer Strain of cyclosporin A with immuno The file of this patent contains at least one drawing Suppressive property and more particularly, to a method of executed in color. Copies of this patent with color drawing producing cycloSporin Aby Submersed fermentation method (s) will be provided by the Patent and Trademark Office comprising the Steps of, Based upon a wild Strain Tolypo upon request and payment of the necessary fee. cladium inflatum which produced a Small amount of FIG. 1A, 1B, and 1C are chromatograms of cyclosporin cyclosporin A, developing amino acid-dependent mutant A, pure product and cycloSporin A produced by Wild Strain Strain induced by UV radiation; conjugating other two KD01 and the high-producing fusant KD461 of Tolypocla amino acid-dependent mutants, making a highly productive 15 dium inflatum, respectively. fusant Strain of cyclosporin A in parallel with increasing FIG. 2 is a photographs showing a colonial morphology demanding of amino acid and organic nitrogen-Source; and of wild strain KD01 and the fusant KD461 of Tolypocladium establishing the Suitable condition and method of culture to inflatum cultured in a malt-yeast extract medium, respec these fusant to obtain the cycloSporin A by Submersed tively. fermentation method. FIG. 3A and 3B are photographs (A) and (B) showing a DESCRIPTION OF THE PRIOR ART morphology of hyphae, culturing wild Strain KD01 and the fusant KD461 of Tolypocladium inflatum in a liquid culture In more detail, Tolypocladium inflatum Gams NRRL medium for 192 hours, respectively. 8044, a producer Strain of cycloSporin A, is fungus and 25 cyclosporin A which this Strain produces is cyclic peptide DETAILED DESCRIPTION OF THE consisting of 11 amino acids and molecular weight is 1,201, INVENTION molecular formula is C2HNO2, and there are 25 The inventors made the targeted fusant with amplified derivatives according to variation of amino acids. Traber cyclosporin A-producing capability, where it comprises, as R., HELVETICA ACTA, 70.13(1987) a method of amplifying production of cycloSporin A, mutat Cyclosporin A, of which chemical name is Cyclo(E)- ing a poor producing Strain of cycloSporin A, wild Strain of (2S, 3R,4R)-3-hydroxy-4-methyl-2-(methyl-amino)-6- Tolypocladium inflatum isolated from Soil making L-valine octenoyl-L-2-aminobutyryl-N-methyl-glycyl-N-methyl-L- dependent mutant and L-leucine-dependent mutant, having leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-O-ala nyl-N- a possibility to be precursor as constituent of target material, methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl), is 35 to amplify dependence on each amino acids, fusing the cells known to have antifungal, antiparasitic and antiinflamma of two amino acid-dependent mutants to increase depen tory properties as well as a potent immunosuppressive dence on both L-valine and L-leucine. property, and is important in the treatment of allograft rejection as well as autoimmune diseases Borel 2 J. F., Prog. Isolation of Cyclosporin A-Producing Wild Strain Allergy 38, 9 (1986)). 40 CycloSporin A-producing wild Strain, Tolypocladium In general, the productive capacity of a producing Strain inflatum KD01, was isolated from soil collected at Mt. Chiri is very important in producing Secondary metabolites by in Chollabuk-do, Korea. fermentation of microorganisms. Soil Sample was cultured on a Solid medium Supple Sesquicillopsis rosariensis G. ARNOLD F605 with 3150 mented with a Small amount of amplicillin to isolate fungi, mg/L and Tolypocladium inflatum Wb6-5 with 1100 mg/L 45 from which family Moniliaceae including Tolypocladium (U.S. Pat. No. 5,256,547, 1993) are known as the highest inflatum was isolated by taxonominical characteristics of productive Strains among known cycloSporin A-producing fungus. The Strain with antifungal property against Aspergil Strains. lus niger was Selected from isolated Strains of family The highly productive CycloSporin A-forming Strain Moniliaceae and then KD01 strain was selected from strains among known Strains has not been reported Since 50 producing cycloSporin A corresponding with its pure product cyclosporin A was for the first time isolated by A. Rueger et by TLC(Thin Layer Chromatography) and HPLC(High Per al. Helv. Chem. Acta 59, 1075 (1976)), in-spite of the fact formance Liquid Chromatography) analysis of their culture that the highly productive mutant of Tolypocladium inflatum extract, identified as Tolypocladium inflatum with charac has been developed actively and used as industrial produc 55 teristics described as table 1 by a classified System of fungus. ing Strain. FIGS. 1A, 1B and 1C chromatograms are presented for cyclosporin A (pure product), cyclosporin A produced by a SUMMARY OF THE INVENTION wild Strain KD01 and the cycloSporin A high producing The inventors made a highly productive cycloSporin fusant, KD-461. In FIG. 1A the cyclosporin A (pure product) A-forming mutant and invented a method for its fermenta 60 is represented by the main peak shown at 4.86 RT, its tion. This mutant is characterized by producing cycloSporin contents being over 98.5%. The cyclosporin A produced in A in high concentration, thus requiring large amounts of the wild Strain KD01 and the cycloSporin A high producing L-Valine and L-leucine as well as organic nitogen Source to fusant, KD-461 are represented in FIGS. 1B and 1C, produce cycloSporin A. respectively, shown at 4.86 RT. The test conditions of the CycloSporin A is cyclic peptide consisting of 11 amino 65 HPLC are those described in Example 1. acids, Valine at position of 5, 11, leucine at position of 4, 6, Isolated wild strain KD01 has low productivity of 175 mg 9, 10 and their derivatives. Tolypocladium inflatum NRRL cyclosporin A per liter. This Strain has corresponding char 5,856,141 3 4 acteristics with Tolypocladium inflatum in terms of follow amino acid-dependent Strains, protoplast of each amino ing; a) slow growth in a malt-yeast extract agar medium, acid-dependent mutant was prepared first by modified b)formation of colony in white and with 5-6 mm of the method of Peberdy et al.(Peberdy, J.
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