Illlllllllllll|||l|l||l||||lllllllllllllll||I||||||||||||||||||ll|lllllllll US0054218'39A United States Patent [191 [11] Patent Number: 5,421,839 Ulbrich et al. [45] Date of Patent: Jun. 6, 1995 [54] ANTIFUNGAL POLYPEPTIDE AND WO89/04371 5/1989 WIPO. PROCESS FOR ITS PRODUCTION OTHER PUBLICATIONS [75] Inventors: Norbert Ulbrich, Berlin; Rolf Applied Microbiology, vol. 13, No. 3, May 1965, pp. Hilgenfeld, Friedrichsdorf; Heinz 314-321; Olson, B. H., et a1.: ‘Alpha sarcin, a new anti Hinel, Obcrursel; Burkhard Sachse, tumor agent’. Kelkheim; Peter Braun, Mainz; Joachim Wink, kodermark; Peter Nucleic Acids Research, 18:13 (1990) Arlington, Va. Eckes, Kelkheim; Jiirgen Logemann; US p. 3987, Wnendt et al.: ‘Cloning and nucleotide Jozef Schell, both of Koln, all of sequence of a cDNA Encoding the antifungal-protein Germany of Aspergillus giganteus and preliminary characterization of the native gene’. [73] Assignee: Hoechst Aktiengesellschaft, .1. Cell. Biochem. Suppl, vol. 14E, 1990, & Symposium Frankfurt am Main, Germany Apr. 16-22, 1990, p. 268, Broglie; R., et al.: Chitinase [21] Appl. No.: 945,982 3 expression in transgenic plants: increased protection against a soil-borne fungal pathogen. ‘ [22] PCT Filed: Jun. 12, 1991 Dur. J. Biochem, vol. 193, No. 1, 1990, pp. 31-38; Na [86] PCT No.: PCT/EP9l/01094 kaya, K., et al.: ‘Amino acid sequence and disul?de § 371 Date: Oct. 30, 1992 bridges of an antifungal-protein isolated from Aspergil lus giganteus’. § 102(e) Date: Oct. 30, 1992 Ar et a1. 1986, Plant Physiol. 81:301-305. [87] PCT Pub. No.: WO91/19738 An. 1985, Plant Physiol 79:568-570. Potrykus, 1991, Annu. Rev. Plant Physiol. Plant M01. PCT Pub. Date: Dec. 26, 1951 Biol. 42:205-225. Primary Examiner-David T. Fox [30]Jun. 15, 1990Foreign [DE] Application Germany Priority...................... Data .. 4o 19 105.2 Assistant Examiner—Erich E. Veitenheimer Mar. 6, 1991 [DE] Germany ...................... .. 41 07 140.9 Attorney, Agent, or Firm—Curtis, Morris & Safford [51] Int. Cl.6 ..................... .. A01B 79/00; AOlN 6100; [57] ABSTRACT 007K 13/00 Antifungal polypeptide and process for its production [52] US. Cl. ......................................... .. 47/58; 514/1; 530/820, 530/823 The polypeptide with the sequence (SEQ ID NO: 1) [58] Field of Search ............... .. 435/69.1, 320.1, 170.1, Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp 435/ 172.3; 800/200, 205, 250, 255, DIG. 43, 44; Asn-Ile-Cys-Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr 47/ 58.03, ‘58.01; 935/ l l, 22; 514/1; 530/820, Ala-Ile-Cys- Lys-Cys-Tyr-Val-Lys-Lys-Cys-Pro-Arg 823 Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly [56] References Cited Lys-Cys-Tyr-Cys us. PATENT DOCUMENTS can be produced by the fermentation of Aspergillus gi ganteus and used as an antifungal agent. 3,104,204 9/1963 Olson . 4,394,443 7/1983 Wcissman et a1. .................. .. 435/68 Expression of this polypeptide gene in plants strongly inhibits the growth of phytopathogenic fungi 0n the FOREIGN PATENT DOCUMENTS plant. ' . 0320130 6/ 1989 European Pat. Off. WO88/00976 2/1988 WIPO . 5 Claims, No Drawings ' 5,421,839 1 2 nitrogenous nutrients: amino acids, peptides and prote ANTIFUNGAL POLYPEPI'IDE AND PROCESS ins, as well as degradation products thereof, such as FOR ITS PRODUCTION peptones or tryptones, and also meat extracts, ground seeds, for example of maize, wheat, beans, soya or the BACKGROUND OF THE INVENTION 5 cotton plant, distillation residues from alcohol produc Antifungal polypeptides, e.g. lectins, have been iso tion, meat meals or yeast extracts, or else ammonium lated from monocotyledonous and dicotyledonous salts and nitrates. The nutrient solution can also contain plants [Ramshaw, J. A. M. (1982) in Nucleic Acids and for example chlorides, carbonates, sulfates or phos Proteins in Plants I. Encyclopedia of Plant Physiology, phates of the alkali metals or alkaline earth metals, iron, New Series, (Boulter, D. and Parthier, B. editors.) vol. zinc and manganese, as additional inorganic salts. 14 A, pp. 229-279, Springer, Berlin]. It has also been The formation of the compound of the polypeptide observed that the biosynthesis of thionine is initiated by according to the invention proceeds particularly well in infestation of the plant with pathogenic fungi. It is a nutrient solution containing 0.5 to 2% of meat ex therefore assumed that this protein possesses antifungal tracts, 0.5 to 2% of peptone, 0.5 to 4% of starch and 0.1 activity [Bohlmann, H. et al. (1989) EMBO J. 7, 15 to 1% of sodium chloride, based in each case on the 1559-1565; Broekaert,>W. F. et al. (1989) Science 245, weight of the total nutrient solution. 1 100-1 102]. The fermentation takes place aerobically, for example Antifimgal polypeptides have also been isolated from submersed with shaking or stirring in shake ?asks or microorganisms. For example, a similar protein has fermenters, with air or oxygen being introduced if ap been detected in the fermentation broth of Aspergillus 20 propriate. It can be carried out over a temperature giganteus (Olson, B. M. & Goerner, G. L. (1965) Appl. range of about 18° to 35° C., preferably at about 25° to Microbiol. 13, 314-321), but the amino acid sequence of 30° C. The microorganism is cultivated under said con said protein is two lysine residues shorter than that of ditions until the stationary phase is reached, i.e. for the protein according to the invention. about 70 to 80 hours. We have now been able to isolate a polypeptide with 25 antifungal action from Aspergillus giganteus and deter The cultivation is advantageously carried out in sev eral stages, i.e. one or more cultures are first mine its sequence. prepared in a liquid nutrient medium and then inocu The nucleotide sequence of the peptide claimed has ,been published by Wnendt et al. (Nucl. Acids Res. 18, p. lated into the actual production medium-the main "3987, September 1990). 30 culture-for example in a volume ratio of 1:10. The We have moreover found that expression, in plants, initial culture is obtained for example by inoculating a of the gene which codes for the polypeptide claimed in sporulated mycelium into a nutrient solution and leav the main patent application cited above strongly inhibits ing it to grow for about 48 to 72 hours. The sporulated the growth of phytopathogenic fungi on the plant. mycelium can be obtained by leaving the strain to grow 35 for about 7 days on a solid or liquid culture medium, for SUMMARY OF THE INVENTION example malt agar. The invention thus relates to: The course of the fermentation can be monitored by A polypeptide with the amino acid sequence (SEQ means of the pH of the culture or the volume of the ID NO: 1) ' mycelium, by thin layer chromatography, by testing the Ala-Thr-Tyr-Asn-G1y-Lys-Cys-Tyr-Lys-Lys-Asp biological activity or by an immunological method. Asn-Ile-Cys-Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr The isolation of the polypeptide from the culture Ala-Ile-Cys- Lys-Cys-Tyr-Val-Lys-Lys-Cys-Pro-Arg medium is effected by known methods, taking into ac Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly count the chemical, physical and biological properties Lys-Cys-Tyr-Cys. of the products. The polypeptide is present in the cul A‘ process for the production of the polypeptide char 45 ture broth. _ acterized above, which process is characterized in that The broth is freed from mycelium and then chro Aspergillus giganteus is cultivated and the polypeptide is matographed on a cation exchanger. After dialysis and ' isolated. concentration, gel ?ltration is carried out, preferably on Use of the Polypeptide as an Antifungal Agent an agarose/acrylamide copolymer. The subsequent A plant cell which expresses the gene of the antifun thorough puri?cation of the polypeptide, which is nec gal peptide from Aspergillus giganteus, as well as a plant essary for the sequence analysis, can be carried out by containing such cells, and plants, plant tissue or plant HPLC using a linear gradient on a column with hydro reproductive material grown from such a cell. phobic interaction. A process for the production of the plant cell, charac The enzymatic digestion of the peptide for elucida terized in that the gene of the antifungal polypeptide 55 tion of the amino acid sequence is carried out by meth from Aspergillus giganteus is expressed in the plant cell. ods known per se and results in the amino acid sequence given above. Heat stability is a characteristic of the DESCRIPTION OF THE PREFERRED enzyme. It shows no loss of activity after heating at 100° EMBODIMENTS C. for a period of 20 minutes. ‘The invention is described in detail below, especially The protein can be used as an antifungal agent, espe . in its preferred embodiments. The invention is further cially against phytopathogenic fungi. The invention determined by the content of the Claims. therefore also relates to a composition for inhibiting or The polypeptide according to the invention is synthe destroying phytopathogenic fungi on plants, which is sized from Aspergillus giganteus with aerobic cultiva characterized in that it contains protein according to the tion. Assimilable carbohydrates and sugar alcohols such 65 invention. Phytopathogens can be successfully con as fructose, lactose or mannitol, and natural products trolled in both a protective and a curative capacity. The containing carbohydrate, are suitable as preferred car spectrum of action of the claimed compound includes a bon sources. The following are possible as preferred large number of different phytopathogenic fungi of 5,421,839 3 4 economic importance, e.g.