AU4418293A - A fungicidally active compound - Google Patents

Description

A FU GICIDALLY ACTIVE COMPOUND

FIELD OF THE INVENTION

The present invention relates to a DNA sequence encoding an antifungal polypeptide, to an antifungal polypeptide encoded by said DNA sequence, to a microorganism and a method for producing the polypeptide, and to a fungicidal composition comprising the antifungal polypeptide. Furthermore, the pre¬ sent invention relates to the use of the polypeptide or the fungicidal composition in combating or controlling fungal at- tack, especially on plants.

BACKGROUND OF THE INVENTION

The fungal species A . giganteus is known to produce an anti¬ fungal protein which is a potential fungicidal agent. Olson and Goerner, 1965, describe the production, isolation and chemical composition of one such antifungal protein from a specific strain of A. giganteus .

Furthermore, Nakaya et al., 1990 and JP-A—4-234399 disclose the amino acid sequence of an A. giganteus antifungal pro¬ tein, Wnendt et al., 1990 the corresponding cDNA sequence, and WO 91/19738 describes the use of this cDNA sequence in the construction of transgenic plants capable of producing the polypeptide. The production and isolation of the anti¬ fungal polypeptide described in these references are reported to have been accomplished by conventional means, i.e. by fer- mentation of an A. giganteus strain capable of and inherently producing the antifungal polypeptide and subsequent recovery of the polypeptide from the fermented culture.

However, the production of the antifungal polypeptide by such procedure is undesirable in that, in general, the yield of antifungal polypeptide is relatively low, and furthermore, in the conventional fermentation, the antifungal polypeptide is produced in combination with and in practice inseparable from a cytotoxic, and thus undesirable component termed α-sarcin, which is frequently produced by strains of A . giganteus .

Accordingly, it would be desirable to be able to produce antifungal A . giganteus polypeptides in larger amounts and at lower costs than what is possible by the above mentioned conventional fermentation, and further to be able to produce antifungal A . giganteus polypeptides essentially free from undesirable A . giganteus components, and in particular free from α-sarcin.

In an abstract from the 23^rd Lunteren Lectures, Jacobs et al. report the expression of the antifungal protein in Saccharo- myceε cereviεiae . Only a very low expression (lng/ml) could be obtained.

Thus, despite the fact that the amino acid and corresponding cDNA sequences of one antifungal A . giganteus polypeptide have been known for several years, it has not, apparently, been found possible to obtain a high recombinant expression thereof.

BRIEF DISCLOSURE OF THE INVENTION

The present inventors have now surprisingly isolated novel antifungal A . giganteus polypeptides (having an amino acid sequence differing from that disclosed in the above cited references) and furthermore succeeded in cloning a gene encoding a novel antifungal polypeptide and obtaining sub¬ stantial recombinant expression from said gene.

Accordingly, in a first aspect the present invention relates to a DNA construct comprising a DNA sequence encoding an antifungal polypeptide, which sequence comprises the nucleo- tide sequence shown in SEQ ID No. 1 or an analogue thereof, which i) hybridizes with a DNA sequence comprising the nucleoti- de sequence shown in SEQ ID No. 1, or with a probe hybridizing with the nucleotide sequence shown in SEQ ID No. 1,

ii) encodes a polypeptide which reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequence shown in SEQ ID No. 2, and/or

iii) encodes a polypeptide having the amino acid sequence shown in the appended SEQ ID No. 2 or a sequence homo¬ logous thereto,

provided that the DNA sequence is different from one which encodes the polypeptide having the amino acid sequence shown in SEQ ID No. 2 except for an asparagine in position 4, an alanine in position 24, a lysine in position 32, a phenylala- nine in position 42 and a tyrosine in position 50.

The DNA sequence excluded in the above mentioned provision encodes the antifungal polypeptide described in WO 91/19738, and an example of such DNA sequence is the cDNA sequence described by Wnendt et al. (1990) .

The properties i)-iii) of the analogous DNA sequence is discussed in detail below.

In a further aspect the invention relates to an antifungal polypeptide comprising the amino acid sequence shown in SEQ ID No. 2, or a variant thereof, which

1) reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequence shown in SEQ ID NO. 2, and/or

2) is homologous to the polypeptide having the amino acid sequence shown in SEQ ID NO. 2, provided that the variant is different from the polypeptide having the amino acid sequence shown in SEQ ID No. 2 except for an asparagine in position 4, an alanine in position 24, a lysine in position 32, a phenylalanine in position 42 and a tyrosine in position 50.

The amino acid sequence excluded by this provision is that of the antifungal protein disclosed in WO 91/19738.

One polypeptide of the invention comprising the amino acid sequence shown in SEQ ID NO. 2 differs from the antifungal protein disclosed in WO 91/19738 as follows:

Asn-4 has been replaced with Pro Ala-24 has been replaced with Gly Lys-32 has been replaced with Arg Phe-42 has been replaced with Leu Tyr-50 has been replaced with His,

the three-letter code of the amino acids being used in its conventional meaning.

Another polypeptide of the invention, being a variant of the polypeptide having the amino acid sequence shown in SEQ ID No. 2, differs from the antifungal protein disclosed in WO 91/19738 as follows:

Asn-4 has been replaced with Asp Lys-32 has been replaced with Val.

It is well-known that any change of the identity of the amino acids present in a protein may have important implications for the structure and activity of the protein, and the above mentioned differences may prove to have important consequen¬ ces for the antifungal activity of the antifungal protein of the invention.

In the present context, the term "antifungal polypeptide" is intended to include the polypeptide comprising the amino acid sequence shown in SEQ ID No. 2 as well as variants thereof as defined herein. The term may be used interchangeably with the term "antifungal protein" and "antifungal peptide". The antifungal polypeptide of the invention is preferably a recombinant polypeptide and as such essentially free from undesirable A . giganteus components. The antifungal polypep¬ tide is preferably substantially pure.

The antifungal activity of the polypeptide (whether of fungi- toxic or fungistatic nature) may be routinely determined by testing the activity of the antifungal polypeptide towards different fungi by well-known procedures. For instance, the antifungal activity (be it quantitative and/or qualitative) may be determined by use of assays as described in Examples 4 and 5 below.

The term "variant" as used in connection with the antifungal polypeptide of the invention is intended to indicate a polypeptide which is derived from the polypeptide having the amino acid sequence shown in SEQ ID NO. 2, or a naturally occurring variant. An example of this latter type of variant is described in Example 1 below. Typically, the variant differ from the native antifungal polypeptide by one or more amino acid residues, which may have been added or deleted from either or both of the N-terminal or C-terminal end of the polypeptide, inserted or deleted at one or more sites within the amino acid sequence of the polypeptide, or sub¬ stituted with one or more amino acid residues within, or at either or both ends of the amino acid sequence of the poly¬ peptide. The variant of the invention has at least one of the characteristic properties l) and 2) discussed in detail below.

In a further important aspect, the present invention relates to a method of producing an antifungal polypeptide, which either comprises the amino acid sequence shown in SEQ ID No. 2 or is a variant of said polypeptide having the properties 1) or 2) defined above, which method comprises

(a) inserting a DNA sequence of the invention encoding the antifungal polypeptide in a suitable expression vector,

(b) transforming a suitable host cell with the recombinant expression vector of step (a) ,

(c) culturing the transformed host cell in a suitable cul¬ ture medium under conditions conducive to the produc¬ tion of the polypeptide, and

(d) recovering the polypeptide from the biomass or culture medium obtained in step (c) .

The method of the invention is believed to be generally applicable for the production of antifungal peptides of the invention as well as of the above mentioned antifungal pro¬ tein described by Nakaya et al. (1990), and is believed to be the first known method resulting in a substantial recombinant production of this type of antifungal peptides, e.g. a yield of more than 200 mg/1.

The antifungal polypeptide obtained by the method has the further advantage of being substantially free from the toxic and thus undesirable compound, α-sarcin, which is frequently produced when native A. giganteus strains or related strains are used as producer organisms.

In a still further aspect the present invention relates to a fungicidal composition comprising, as an active ingredient, an antifungal polypeptide of the invention or an antifungal polypeptide produced by the method of the invention. The fungicidal composition further contains a suitable excipient.

Within the context of the present invention a method of combating or controlling fungi at a locus infested or liable to be infested with a fungus is envisaged, which method comprises applying to said locus an antifungal polypeptide of the invention, a fungicidal composition of the invention, or a microorganism of the invention capable of producing the antifungal polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The DNA construct of the invention

In the DNA construct of the invention, the analogue of the DNA sequence shown in SEQ ID No. 1 may, for instance, be a subsequence of said DNA sequence, a genetically engineered modification of said sequence which may be prepared by well- known procedures, e.g. by site-directed mutagenesis, and/or a DNA sequence isolated from another organism and encoding an antifungal polypeptide. In any event the analogue should have at least one of the properties i)-iii) listed above. These properties are further discussed below.

Property i) , i.e. the capability of the analogous DNA sequen¬ ce of hybridizing with either a DNA sequence comprising the DNA sequence SEQ ID No. 1, or with a probe hybridizing with the DNA sequence shown in SEQ ID No. 1, is intended to be understood broadly. Thus, the hybridization may be performed under any suitable conditions known in the art conveniently used to assess hybridization, e.g. as described by Sambrook et al. (1989) . As an example the hybridization may be deter¬ mined by a method which involves presoaking in 5xSSC and prehybridizing for 1 h at -40°C in a solution of 5xSSC,

5xDenhardt^,s solution, 50 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybri¬ dization in the same solution supplemented with 50 μCi 32-P- dCTP labelled probe for 18 h at -40°C followed by washing three times in 2xSSC, 0.2% SDS at 40°C for 30 minutes.

Property ii) , i.e. the immunological cross reactivity may be assayed using an antibody raised against or reactive with at least one epitope of the antifungal A . giganteus polypeptide having the amino acid sequence shown in SEQ ID No. 2. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. In the Materials and Methods section below, a convenient method for producing antibodies is desc¬ ribed (vide the section titled "Production of antibodies against the antifungal protein") . The immunological cross- reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodif- fusion assay, e.g. as described by Hudson et al., 1989.

The ho ology between polypeptides encoded by the DNA con¬ struct of the invention and the amino acid sequence shown in SEQ ID No. 2 (i.e. property iii) above) is intended to denote a degree of identity between the two sequences indicating a derivation of the first sequence from the second. In particu¬ lar, a polypeptide is considered to be homologous to the antifungal polypeptide encoded by the DNA construct, if a comparison of the amino acid sequences of the polypeptides reveals an identity of greater than about 70%, such as grea- ter than about 80%, 85%, 90%, 92%, 94%, 96% or even as high as 98%. Sequence comparisons can be performed via known algorithms, such as the one described by Lipman and Pearson (1985) .

It is well known that homology exists between polypeptides of different origins and it is contemplated that in the DNA construct of the invention, the DNA sequence encoding an antifungal polypeptide may be derived from an animal inclu¬ ding a mammal, an insect, a plant, a protozoae, an algae, or a microorganism.

In the present context, especially interesting origins are bacteria and fungi. The term "fungi" is intended to include yeasts and filamentous fungi. As stated above the DNA sequen¬ ce shown in SEQ ID No. l was derived from a strain of the fungal genus Aεpergillus, more specifically from a strain of A . giganteus . DNA sequences encoding antifungal polypeptides being identical to or variants of the antifungal polypeptide comprising the amino acid sequence shown in SEQ ID No. 2 may be obtained from strains of other Aspergillus spp., such as A. pallidus, A. clavatuε , A. longiveεica , A. rhizopoduε or A. 5 clavatonanicuε, because of the close relationship which exists between these species and A . giganteuε . In fact, an antifungal polypeptide comprising the amino acid sequence shown in SEQ ID No. 2 was obtained from A . clavatonanicuε and a variant of said polypeptide (further described in Example 101) was obtained form A. pallidus and A. clavatuε .

A DNA sequence of the invention encoding an antifungal poly¬ peptide may be isolated by well-known methods. Thus, the DNA sequence may, for instance, be isolated by establishing a 15 cDNA or genomic library from an organism expected to harbour the sequence, especially a microorganism as defined above, and screening for positive clones by conventional procedures.

Examples of such procedures are hybridization to oligonucleo¬ tide probes synthesized on the basis of the full or partial

20 amino acid sequence of an antifungal polypeptide as disclosed herein (e.g. on the basis of the amino acid sequence shown in SEQ ID No. 2) or on the basis of the DNA sequence shown in SEQ ID No. 1; selection for clones expressing the appropriate antifungal activity; or selection for clones producing a

25 polypeptide which is reactive with an antibody raised against an antifungal polypeptide, e.g. using the techniques descri¬ bed in the section "Materials and Methods" below.

A preferred method of isolating a DNA sequence from a cDNA or genomic library is by use of polymerase chain reaction (PCR) 30 using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence of an antifungal polypeptide and/or a DNA sequence encoding such. For instance, the PCR may be carried out using the techniques described in US 4,683,202 or R.K. Saiki et al. (1988).

35 Alternatively, the DNA sequence encoding an antifungal poly- peptide may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage et al., (1981) or the method described by Matthes et al. (1984) . According to the phosphoamidite method, oligonucleo- tides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vec¬ tors.

Finally, the DNA sequence encoding an antifungal polypeptide may be of mixed genomic and synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropria¬ te) , the fragments corresponding to various parts of the entire DNA sequence in accordance with standard techniques.

The antifungal polypeptide of the invention Properties 1) and 2) characterizing variants of the anti¬ fungal polypeptide comprising the amino acid sequence shown in SEQ ID No. 2 are intended to be understood in a similar manner to properties ii) and iii) defined above in connection with the DNA construct of the invention.

Furthermore, it will be understood that an antifungal poly¬ peptide of the invention is obtainable from the same sources as the DNA sequence encoding it. Thus, it is contemplated that the antifungal polypeptide of the invention is obtai¬ nable from any of the above specified organisms.

In this connection, the antifungal A . giganteus polypeptide of the invention having the amino acid sequence shown in SEQ ID NO. 2 has been isolated from different strains of the fungal species A. giganteus , cf. Example 1 hereinafter, and from a strain of A . clavatonanicuε .

As explained above the variant of the antifungal polypeptide of the invention may be a naturally derived variant, i.e. a polypeptide isolated from any of the above mentioned orga¬ nisms or encoded by a DNA sequence isolated or produced from any of the organisms. An example of such variant is one which differ from the amino acid sequence shown in SEQ ID No. 2 by having an Asp in position 4, an Ala in position 24, a Val in position 32, a Phe in position 42 and a Tyr in position 50. Alternatively, the variant may be a synthetically produced variant constructed on the basis of the knowledge of the amino acid sequence of an antifungal polypeptide of the invention.

It is contemplated that interesting modifications of the antifungal polypeptide of the invention, and thereby inter¬ esting variants, may be identified on the basis of an analy¬ sis of the conformational structure and the active site of the antifungal polypeptide using techniques known in the art, examples of which are NMR (e.g. as described in Protein Engineering, 1987) , X-ray diffraction, computer modelling and the like. When one or more interesting modifications of the amino acid sequence of the antifungal polypeptide have been identified, the corresponding variants may be prepared by suitably modifying a nucleotide sequence encoding the anti- fungal polypeptide.

For instance, a genetically modified variant of an antifungal polypeptide of the invention may be prepared, by suitably modifying a genomic or cDNA sequence comprising the DNA sequence shown in SEQ ID No. 1 at a site corresponding to the site(s) at which it is desired to introduce amino acid sub¬ stitutions. A suitable method for performing such modifica¬ tion is site-directed mutagenesis using synthetic oligonucle- otides encoding the desired amino acid sequence for homolo¬ gous recombination in accordance with well-known procedures. Alternatively, random mutagenesis, using e.g. radiation or chemical treatment, may be employed. Furthermore, variants may be prepared by chemical modification of the antifungal peptide produced in accordance with the invention. The method of the invention for producing an antifungal polypeptide

In the method of the invention for the recombinant production of an antifungal polypeptide, the DNA sequence encoding the antifungal polypeptide may be one isolated or constructed as described above, and is preferable one derived from a cDNA or genomic library. The antifungal polypeptide to be produced by the method may be an antifungal polypeptide of the invention or the antifungal protein described by Nakaya et al. (1990) .

The host cell used for the production of the antifungal polypeptide may be any cell which is capable of producing the antifungal polypeptide, and any cell, the growth of which is not subjected to any substantial or total inhibition by the antifungal polypeptide. The host cell may be a higher eu- karyotic cell such as an insect cell or a prokaryotic or a eukaryotic microorganism, such as a bacterium or a fungus.

Fungal host cells of particular interest is filamentous fungi. The term "filamentous fungi" is intended to include fungi belonging to the groups Phycomyceteε , Zygomyceteε, As corny ceteε , Baεidiomyceteε or fungi imperfecti, including Hyphomyceteε such as the genera Aεpergilluε , Trichoderma , Penicillium , Neuroεpora or Humicola . In particular, strains of Aεpergilluε oryzae are considered to be suitable host cells (although such cells may be sensitive as such to the antifungal polypeptide) , in that the antifungal protein of the invention has been found not to have an inhibitory ac¬ tivity substantial enough to prevent production in strains of this species.

The use of Aεpergilluε spp. for the expression of proteins is described in, e.g., EP 272 277 and EP 238 023.

Alternatively, yeast cells or bacterial cells may be used as host cells. Examples of suitable yeast cells include cells of Saccharomyceε spp. or Schizoεaccharomyceε spp., and cells of Klyveromyceε lactiε , Yarrowia lipolytica , Pichia pastoris and Hanεenula poly orpha . Examples of suitable bacterial cells include cells of Bacillus spp., such as of B . εubtiliε .

When the antifungal protein described in Nakaya et al. (1990) is to be produced, the host cell is preferably a bacterial cell or a cell of a filamentous fungus.

The recombinant expression vector into which the DNA sequence is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously repli¬ cating vector, i.e. a vector which exists as an extrachromo- so al entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The recombinant DNA vector should further comprise DNA sequences encoding functions permitting gene expression, and where appropriate a suitable marker for the selection of transformants and/or a preregion allowing the secretion of the expressed protein in a correctly processed form from the host cell.

DNA sequences encoding functions permitting gene expression typically comprise a promoter, transcription initiation sites, and transcription termination and polyadenylation functions.

The promoter which may be preceded by upstream activating sequences and enhancer sequences as known in the art may be any DNA sequence exhibiting a strong transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al. (1980)), (Alber and Kawasaki (1982)), (Young et al. (1982)) or the TPI1 (US 4, 599, 311) or ADH2-4C (Russell et al., Nature 304, 1983, pp. 652-654) promoters. Suitable promoters for use in filamentous fungus host cells are, for instance, the A . nidulanε ADH3 promoter (McKnight et al., (1985) the tpiA promoter, or promoters derived from the gene encoding A . oryzae TAKA amylase, A . niger neutral α-amylase, A . niger acid stable α-amylase, A . niger glucoamylase, A . oryzae alkaline protease or A . oryzae triose phosphate isomerase.

In particular when the host organism is A . oryzae , a prefer¬ red promoter for use in the process of the present invention is the A . oryzae TAKA amylase promoter as it exhibits a strong transcriptional activity in A . oryzae . The sequence of the TAKA amylase promoter appears from EP 238 023.

Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The preregion provided on the vector to ensure efficient direction of the expressed product into the secretory pathway of the host cell and subsequent processing may be a naturally occurring signal or leader peptide or a functional part thereof or a synthetic sequence providing secretion of the protein from the cell.

In particular, when the host cell is a filamentous fungus, the preregion may be derived from or be a gene encoding an Aεpergilluε sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, a gene encoding a Coprinus sp. peroxidase, a gene encoding a Humicola cellula- se, lipase or xylanase, e.g. the gene encoding A . oryzae TAKA amylase, A . niger neutral α-amylase, A . niger acid-stable α- amylase, A . niger glucoamylase, or a Coprinus macrorhizuε or cinereuε peroxidase, H . lanuginoεa lipase, H . inεolenε cellu- lase or xylanase. Furthermore, the preregion may be the one which, in nature, is associated with the DNA encoding the antifungal peptide of the invention.

In the method of the present invention, the host cell may be transformed with a vector comprising a DNA sequence coding for a selection marker which is capable of being incorporated in the genome of the host organism on transformation, but which is either not expressed by the host before transfor¬ mation or expressed in amounts which are not sufficient to permit growth under selective conditions. Transformants can then be selected and isolated from non-transformants on the basis of the incorporated selection marker.

Suitable selection markers may be derived, e.g. from the A. nidulanε or A . niger argB gene, the A . nidulanε trpC gene, the A. nidulanε amdS gene, the A . nidulanε εC gene, the Neuroεpora craεsa pyr4 or DHFR genes, or the A . niger or A . oryzae niaD gene.

The procedures used to ligate the DNA sequences encoding an antifungal polypeptide of the invention, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for in¬ stance, Sambrook et al.,1989).

The techniques used to transform a fungal host cell may suitably be adapted from the methods of transforming A. nidulanε described in, for instance, Yelton et al., Proc. Natl. Acad. Sci. USA 81. 1984, pp. 1470-1474, or EP 215 594, or from the methods of transforming A . niger described in, for instance Buxton et al. (1985) or US 4,885,249, or from the method of transforming A. oryzae described in EP 238 023.

In addition to the above described preferred recombinant production method an antifungal polypeptide of the invention may be produced by a conventional method, comprising cultiva- ting a microorganism which, in nature, is capable of pro¬ ducing the antifungal polypeptide, e.g. a strain of Aεper¬ gilluε and in particular of the species A . giganteuε, on a suitable culture medium and under conditions allowing the production of the polypeptide, and recovering the polypeptide the resulting biomass and/or fermented culture medium.

Irrespective of the production method used, the term "sui¬ table culture medium" is intended to indicate a medium which contains the micro- and macronutrients required for obtaining a satisfactory growth of the microorganism and a satisfactory production of an antifungal polypeptide of the invention. Thus, the culture medium typically comprises sources of carbon and nitrogen assimilable by the microorganism and generally low levels of inorganic salts and trace metals. Specific examples of a suitable culture media are given in the Materials and Methods section below.

Suitable cultivation conditions allowing the production of an antifungal polypeptide as described herein are, inter alia, submerged, aerobic growth in shake flasks or fermenters.

The main part of the antifungal polypeptide is preferably excreted into the fermentation medium (as a consequence of being associated with a suitable pre-region) and may be recovered from the medium by any suitable means. One con¬ venient method involves subjecting the medium to a cation exchange chromatography treatment followed by ultrafiltra- tion, and optionally, if required, repeating this treatment one or more times.

When a fungal cell is used for the production of an anti¬ fungal polypeptide, the mycelium resulting from the cultiva- tion may harbour a minor amount of the polypeptide and thus, it may be desirable to isolate the polypeptide from the myce¬ lium. This isolation is conveniently accomplished by extrac¬ ting the polypeptide from the mycelium with a suitable sol¬ vent or buffer and, after removal of the mycelium, subjecting the extracted polypeptide present in the solvent or buffer to the above described recovery method, either alone or in combination with the fermentation medium.

The mycelium is normally removed from the fermentation medium and/or extraction buffer by centrifugation and/or filtration.

The purity of the isolated polypeptide may be determined by use of well-known methods such as HPLC.

Once recovered, the antifungal polypeptide may be subjected to a suitable modification, e.g. an enzymatic or chemical modification, in order to obtain a modified polypeptide having improved properties such as an improved antifungal activity.

In a further important aspect the present invention relates to a fungicidal composition comprising, as an active ingredient, an antifungal polypeptide of the invention.

Alternatively or additionally, the fungicidal composition may comprise a microorganism capable of expressing an antifungal polypeptide of the invention, or a fermented culture medium obtained by fermentation of the microorganism. In this re- spect it is preferred that the microorganism is prepared by recombinant DNA techniques.

The fungicidal composition of the invention may for agronomi¬ cal and/or horticultural applications be formulated by mixing the active principle with suitable inert and compatible carriers or diluents to obtain a composition of the type generally used in agricultural compositions such as a wettable powder, an emulsifiable concentrate, a concentrated emulsion, a granular formulation, a water soluble powder, a solution, a suspension concentrate, a release formulation, an alginate, a xanthan gum and/or an aerosol. As solid carriers bentonite diatomaceous earth, apatite, gypsum, talc, pyrophyllite, vermiculite, ground shells, and clay may be mentioned. A surface active agent may also be added with the purpose of producing a homogeneous and stable formulation.

The diluent or carrier in the composition of the invention can as indicated be a solid or a liquid optionally in asso- ciation with a surface-active agent, for example a dispersing agent, emulsifying agent or wetting agent. Suitable surface- active agents include anionic compounds such as a carboxy¬ late, for example a metal carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate or sodium cetyl sulphate; ethoxy- lated fatty alcohol sulphates; ethoxylated alkylphenol sul¬ phates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzene sulphonates or lower alkyl- naphthalene sulphonates, e.g. butyl-naphthalene sulphonate; salts of εulphonated naphthalene-formaldehyde condensates; salts of sulphonated phenol-formaldehyde condensates; or more complex sulphonates such as the amide sulphonates, e.g. the sulphonated condensation product of oleic acid and N-methyl taurine or the dialkyl εulphosuccinateε, e.g. the sodium sul¬ phonate of dioctyl succinate. Nonionic agents include conden¬ sation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- of alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g. sorbitan fatty acid esters, condensation pro¬ ducts of such esters with ethylene oxide, e.g. polyoxy- ethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.

Examples of a cationic surface-active agent include, for in¬ stance, an aliphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate; an oxygen-containing amine such as an amine oxide or polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

The composition of the invention may be in any form known in the art for the formulation of agrochemicals, for example, a solution, a dispersion, an aqueous emulsion, a dusting pow- der, a seed dressing, a dispersible powder, an emulsifiable concentrate or granules. Moreover, it can be in a suitable form for direct application or as a concentrate or primary composition which requires dilution with a suitable quantity of water or other diluent before application.

An emulsifiable concentrate comprises the active ingredient dissolved in a water-immiscible solvent which is formed into an emulsion with water in the presence of an emulsifying agent.

A dusting powder comprises the active ingredient intimately mixed and ground with a solid pulverulent diluent, for example, kaolin.

A granular solid comprises the active ingredient associated with similar diluents to those which may be employed in dust¬ ing powders, but the mixture is granulated by known methods. Alternatively it comprises the active ingredient absorbed or adsorbed on a pre-granular diluent for example, Fuller's earth, attapulgite or limestone grit.

Wettable powders, granules or grains usually comprise the active ingredient in admixture with a suitable surfactant and an inert powder diluent such as china clay.

Another suitable concentrate is a flowable suspension con¬ centrate which is formed by grinding the active ingredient with water or other liquid, a wetting agent and suspending agent.

The concentration of the antifungal polypeptide in the compo¬ sitions of the invention may vary within a wide range depending on the type of formulation and the field of appli¬ cation.

It is contemplated that the active compound of the invention may be applied in concentrations ranging from about 0.01 mg/ml to 10 mg/ml, preferably from 0.05 mg/ml to 1 mg/ml, and in particular about 0.1 mg/ml for use in controlling fungi in plants.

Depending on the circumstances such as the crop wherein fungi are to be combated, the environmental conditions or other factors, a composition of the invention may, in addition to an antifungal polypeptide of the invention, also contain other active ingredients such as other biocides, e.g. fungi¬ cides, pesticides, herbicides, insecticides, nematocides, or acaricides, or plant growth regulators such as plant nutri- ents or fertilizers.

Examples of other fungicides which can be combined with an antifungal polypeptide of the invention include especially ergosterol biosynthesis inhibitors ("EBIs") . These are ge¬ nerally imidazole or triazole derivatives and examples in- elude those known by the common names prochloraz, triadime- fon, propiconazole, diclobutrazol, triadiminol, flusilazole, flutriafol, myclobutanil, penconazole, quinconazole, imazalil and diniconazole. Examples of non azole Ebis include nuari- mol, fenari ol, fenpropimorph, tridemorph and fenpropidine.

Other fungicides which can be combined with an antifungal polypeptide of the invention include:

Dithiocarbamates, e.g. thiram, maneb, zineb and mancozeb; Phatalimides, e.g. captan, folpet and captafol; Carboxines, e.g. carboxin and oxycarboxin; Benzimidazoles, e.g. benomyl, carbendazim and fuberidazole; Carbamates, e.g. prothiocarb and propamocarb; Isoxazoles, e.g. hymexazol; Cyanoacetamides, e.g. cymoxanil; Ethylphosphonates, e.g. fosetylalu inium;

Phenylamides, e.g. furalaxyl, metalaxyl, benalaxyl, of race, cyprofuram and oxandixyl;

Dicarboximides, e.g. procymidone, iprodione and vinclozolin; Organophosphorous fungicides, e.g. pyrazophos, triamiphos, ditalimfos and tolcofosmethyl; and

Aromatic hydrocarbon fungicides, e.g. quintozene, dichloren, and diphenyl.

In a further aspect the present invention relates to a method of combating or controlling fungi at a locus infested or liable to be infested with a fungus, comprising applying to said locus an antifungal polypeptide of the invention, a fungicidal composition of the invention, a fermented medium comprising an antifungal polypeptide of the invention, or a microorganism capable of producing an antifungal polypeptide of the invention.

The antifungal polypeptide of the invention has been found to be particularly potent against fungi belonging to the class Ascomycetes, Deuteromycetes, or Oomycetes and accordingly the locus to be treated is one susceptible to attack by such fungi.

In particular, the antifungal polypeptide has been found be highly effective for the control of fungi belonging to the genus, Botrytiε , especially B . cinerea , the genus Pyrenophora , especially the species P. teres , the genus Phoma , especially the species P. lingam , or the genus Cochlioboluε , especially the species C. sativuε . The anti¬ fungal protein of the invention was surprisingly found to have another activity spectrum than that reported for the antifungal protein disclosed in WO 91/19738.

Normally, the locus to be treated is a plant or a plant part such as foliage, seeds, tubers, cuttings etc., or soil to be used for growing plants, and the like. In this connection an antifungal polypeptide or a composition of the invention may for agronomical or horticultural uses be applied to a region to be treated either directly to the soil as a pre-emergence treatment or to the foliage or fruits of the plants as a post-emergence treatment. Depending on the crop and circumstances the treatment may be postponed until seeds or fruits appear on the plants, wherein fungi are to be controlled.

An antifungal polypeptide or a composition of the invention can be applied directly to the plant or plant part by, for example, spraying or dusting either at the time when the fungus has begun to appear on the plant or before the appe¬ arance of fungus as a protective measure or as a combination of both, i.e. prophylactic and curative. In all three cases the preferred mode of application is by foliar spraying.

Sometimes, it is practicable to treat the roots of a plant before or during planting, for example by dipping the roots in a suitable liquid or solid composition. When an antifungal polypeptide or a composition of the invention is applied directly to the plant a suitable rate of application is from 0.005 to 5 kg per hectare, preferably from 0.01 to 2 kg per hectare of the active ingredient.

In this method of the invention the active ingredient may, alone or in combination with a conventional biocide, also be applied to seeds or habitat. Thus the active ingredient or the composition comprising it can be applied directly to the soil before, at or after drilling so that the presence of active ingredient in the soil can control the growth of fungi which may attack seeds.

The antifungal polypeptide or the composition may be applied in amounts corresponding to from about 5 g to about 5 kg of the active ingredient per hectare. When the soil is treated directly the active ingredient alone or in admixture with the conventional biocide can be applied in any manner which allows it to be intimately mixed with the soil such as by spraying, by broadcasting a solid form of granules, or by applying the active ingredient at the same time as drilling by inserting it in the same drill as the seeds. A suitable application rate is within the range of from 0.005 to 5 kg per hectare, more preferably from 0.05 to 2 kg per hectare.

The concentration of antifungal polypeptide in a composition of the invention when used alone or in combination with a conventional fungicide, as applied to plants is preferably within the range from about 0.001 to about 30 per cent by weight, especially 0.01 to 3.0 per cent by weight. In a primary composition the amount of the active ingredient can vary widely and can be, for example, in the range from about 5 to about 95 per cent by weight of the composition.

The concentration of the other fungicidally active ingredient in the mixed composition of the present invention, as applied to plants is preferably within the range of 0.001 to 50 per cent by weight, especially 0.01 to 10 per cent by weight. In a primary composition the amount of such other active ingre- dients can vary widely and can be, for example, from 5 to 80 per cent by weight of the composition.

Although the present invention has been described in detail in connection with controlling fungi in plants, it is anti¬ cipated that an antifungal polypeptide or a composition of the invention may be used for combating or controlling fungi in general, especially fungi belonging to the class Aε corny ceteε , Deuteromyceteε, or Oomyceteε and more particu¬ larly fungi belonging to the genus Botrytiε, especially the species B . cinerea , the genus Pyrenophora , especially the species P . tereε, the genus Phoma , especially the species P. lingam , or the genus Cochlioboluε , especially the species C. εativuε .

Examples of uses of an antifungal polypeptide of the inven¬ tion are for the preservation of wood by adding said com- pounds to wood preservation and/or impregnation compositions, and as a fungicide and preservant in paints - both to prevent growth in the paint during storage, and growth on the painted object such as the plastered surface of a house.

5 Further when an antifungal polypeptide of the invention is produced by recombinant DNA techniques it is contemplated to be used for the preservation of foods and feeds and for treating mammals, including humans, and non-mammalian ani¬ mals. More specifically, the antifungal polypeptide may be 0 used in the preparation of a veterinarian or human medicament for the treatment of fungal infections, and may accordingly be a constituent in such medicament, e.g. in a concentration of 0.001-1 mg/ml.

Also, fungicidally active compounds of the invention may be 15 used as fungicidal additive to growth media for various microorganisms such as E . coli and Pseudomonaε aeroginoεa .

The present invention is further illustrated in the following examples which are not intended to limit, in any way, the scope of the invention as claimed.

20 EXAMPLES

MATERIALS AND METHODS

A. giganteus strains

The A . giganteuε strain deposited with the Centraalbureau voor Schimmelculturen (CBS) under the accession number CBS 25526.65 (referred to as A3274) was used for the experiments together with another A . giganteuε strains termed A3273.

Furthermore, a strain of A . palliduε (A3271) , two strains of

A . clavatonanicus (A3275 and A3276) , and a strain of A. clavatuε (A3270) were used for the production of antifungal 30 peptides of the invention. Cultivation of an A. giganteus strain producing the anti¬ fungal protein

Cultivation on agar εlantε was carried out using an agar medium prepared from 39 g of Potato Dextrose Agar (from 5 Difco) and distilled water up to 1000 ml. The agar slants was inoculated with the A. giganteuε strain and grown for one week at a 26°C.

Submerged cultivation

A 500 ml Erlenmeyer flask containing 100 ml of AMC substrate

10 (15 g of meat extract, 20 g of Peptone, 20 g of corn starch, 5 g of NaCl, 1 ml of Pluronic, 1 1 of H₂0) was inoculated with 5 ml of a spore suspension containing 10⁶ spores/ml prepared from an agar slant culture of A . giganteuε obtained as described above containing 10⁶ spores per ml. The flask

15 was shaken at 220 rpm at 30°C for 3 days after which the antifungal protein could be recovered.

Isolation, purification and amino acid sequence determination of the antifungal protein

The fermentation broth obtained as described above was sub-

20 jected to centrifugation, the mycelium was suspended in Tris buffered saline at pH 7 and then subjected to a second cen¬ trifugation. The supernatants from the two centrif gations were combined and subjected to sterile filtration. The pH of the resulting supernatant preparation was adjusted to between

256.5 and 9 (the actual value being indicated in the respective examples) and the supernatant preparation was applied to a cation exchange resin, S Sepharose Fast Flow, which prior to application was equilibrated with a phosphate buffer at pH 6.5. Elution of active fractions from this resin was

30 accomplished by the application of a buffer with a high ionic strength such as a buffer comprising 20 mM phosphate, 1.5 M NaCl, pH 6.5. If necessary, the procedure was repeated after dilution with or dialysis against a buffer with ionic strength below or near that of 10 mM phosphate at pH 6.0. Elution from this second step may, if necessary, be carried out as a gradient elution. The purity of the active fractions was assessed by HPLC.

The combined active fractions were subjected to sterile filtration in a 0.22 μm filter (Millipore) prior to testing of the antifungal properties of the purified protein.

The purified antifungal protein was S-carboxymethylated using the method described by Nakaya et al. 1990 and desalted using reversed phase HPLC. The S-carboxymethylated antifungal protein was subjected to N-terminal amino acid sequence determination on an Applied Biosystems 473A sequencer operated in accordance with the manufacturers instructions.

Extinction coefficient

The extinction coefficient of the antifungal protein was estimated from the amino acid sequence shown in SEQ ID NO. 2 using the formula

ε^01%(280nm.=5690- CNo. Trp)+1280• (No. Tyr)+120• (No. Cys) molecular mass

(Gill & von Hippel, 1989), where (No. Trp), (No. Tyr) and (No. Cys) are the number of Trp, Tyr and Cys residues in the amino acid sequence.

Based on this formula the extinction coefficient at 280nm has been calculated to 1.3. All protein determinations are based on OD₂₈₀ measurements using this calculated extinction co- efficient. DNA synthesis

DNA synthesis (e.g. nucleotide probes) was carried out using an Applied Biosystems Model 380A DNA synthesizer.

PCR amplification Unless otherwise stated PCR amplification was carried out in accordance with the standard PCR protocol described by, e.g., Innis and Gelfand, 1990) .

Production of antibodies against the antifungal protein The antifungal peptide produced and purified as described above was coupled to cationized Bovine Serum Albumin (BSA) according to the procedure described by Pierce, kit no. 77151. More specifically, 2 mg cationized BSA was solubilized in 200 μl Milli Q water. The antifungal peptide was diluted to 1.0 mg/ml in 0.1 M MES [2-N-Morpholino-ethanesulfonic acid] containing 0.9 M NaCl, pH 4.7. 10 mg EDC [l-ethyl-3-(3- Dimethylaminopropyl)-carbodiimide] were dissolved in 1 ml Milli Q water. A reaction mixture consisting of 200 μl BSA, 1400 μl antifungal peptide and 100 μl EDC was prepared and the reaction continued for 2 h at ambient temperature. The coupled protein was desalted on a gelfiltration column e- quilibrated with 80 mM sodium phosphate containing 0.9 M NaCl, pH 7.2 and further diluted to a concentration of 25-250 μg/ml.Rabbits were immunized with the purified protein in Freund's complete adjuvant by use of an immunization scheme essentially according to the standard procedures described by Hudson et al., 1989. Serum may be collected and tested for polyspecific antibodies against the antifungal protein in a single radial im unodiffusion assay or a western blot assay using the purified antifungal protein obtained as described above as an antigen. The assays may be carried in accordance with standard procedures, e.g. as described by Hudson et al., 1989. The antibodies may be purified by standard methods such as standard immunoadsorbent techniques using the purified antifungal protein immobilized to a carrier, e.g. as descri- bed by Hudson et al., 1989. EXAMPLES

EXAMPLE 1

Production of antifungal polypeptides of the invention A) The A . giganteuε strains A3273 and A3274, respectively, were cultivated under a submerged cultivation as described above, and an antifungal protein of the invention was isola¬ ted and purified by the procedures outlined above with the modifications mentioned below.

A3273 The pH of the supernatant preparation applied to the cation exchange resin was adjusted to pH 6.5.

The HPLC analysis of the eluate, obtained from the purifica¬ tion of the supernatant preparation on the S Sepharose Fast Flow cation exchange resin, resulted in two peaks which had substantially different retention times. The amino acid sequencing of the protein of the first peak showed that the protein was identical with the corresponding part of the antifungal protein, the amino acid sequence of which is shown in WO 91/19738, except for the amino acid residues 4 (Asn to Pro) , 24 (Ala to Gly) , 32 (Lys to Arg) , 42 (Phe to Leu) , and 50 (Tyr to His) .

Of the first 38 amino acid residues of the protein of the second peak 35 residues (92%) were identical with the cor¬ responding residues of alpha sarcin from A. giganteuε (Sacco et al. , 1983) .

The first S Sepharose eluate was purified further on a S Sepharose Hiperformance Hiload column by gradient elution resulting in the recovery of a more than 95% pure antifungal protein as assessed by HPLC.

By amino acid sequencing as described in Materials and Methods above, the purified protein was found to have the amino acid sequence shown in SEQ ID NO. 2.

The purified A3273 antifungal protein was mixed with a buffer containing 0.2 M phosphate, pH 6.0, and 0.3 M NaCl and stored at -20°C. The optical density (OD) was determined to 0.8 on at 280 nm, corresponding to a concentration of about 0.5 mg of the protein per ml. This concentration was estimated to be about 20 times higher than the concentration of the protein in the fermentation medium.

A3274 The antifungal protein of A3274 was purified by the above described procedure with the exception that the eluate from the last step was diluted to a resulting buffer (20 mM phos¬ phate, 100 mM NaCl, pH 7) in order to obtain a lower ionic strength in the final product.

A total of 1.6 litre of fermentation broth, obtained as described in the Materials and Methods section above under "Submerged cultivation", were subjected to purification resulting in a yield of the purified A3274 antifungal protein of about 20 mg.

The purification from A3274 revealed that A3274 produces a lower amount of the antifungal protein than A3273 and that the ratio between the amount of the antifungal protein and the alpha sarcin is higher in A3274 than in A3273. This observation presumably reflects strain differences.

The purity of the purified A3274 antifungal protein was higher than 95% as assessed by a HPLC analyses as described above.

By sequencing as above, the amino acid sequence of the A3274 antifungal protein was shown to be identical to that of the A3273 protein, the sequence of which is shown in SEQ ID NO. 2. Thus, the antifungal protein of the invention have been found to be produced by two different A . giganteuε strains. The purified A3274 antifungal protein was diluted against a buffer containing 20 mM phosphate and 100 mM NaCl, pH 7.0, and stored at -20°C. The optical density at 280 nm (OD280) was determined to 1.3 corresponding to a the concentration of protein to about 1.0 mg per ml (assuming the extinction coefficient e (280nm)=1.3) . This preparation was used in the in vitro assay described in Example 4.

By mass spectroscopy analysis of the antifungal protein the molecular weight was confirmed to be the one expected from the amino acid sequence shown in SEQ ID NO. 2, namely 5735 Da.

In a similar manner to that described above, antifungal peptides were isolated from the A . clavatonanicuε strains A2375 and A3276 and subsequently characterized. The anti- fungal peptides produced by these strains were found to have the amino acid sequence shown in SEQ ID No. 2.

A3270 and A3271

The two Aεpergilluε strains A . clavatuε (A3270) and A . palli- duε (A3271) were submerged cultivated and purified as described in Materials and Methods. HPLC analyses of the eluate obtained from the S Sepharose column revealed two peaks with substantially different retention times. Amino acid sequencing of the first peak eluting with a retention time similar to the antifungal peptide obtained as described above showed that the peptide was identical with the the antifungal peptide described in WO 91/19738, except for the amino acid residues 4 (Asn to Asp) and 32 (Lys to Val) .

EXAMPLE 2

Cloning of a gene encoding an antifungal A . giganteuε poly- peptide

On the basis of the amino acid sequence of the antifungal A . giganteuε polypeptide shown in SEQ ID No. 2 and of the cDNA sequence published by Wnendt et al. (1990) encoding a different antifungal polypeptide, the following primers were synthesized:

1) Two long, non-degenerate oligomers having the following DNA sequences:

57 mer: Oligo 4641 (SEQ ID No. 3): GGCAAATGCTACAAGAAGGATAATATCTGCAAGTACAAGGCACAGAGCGGCAAGACT 3'

50 mer: Oligo 4642 (SEQ ID No. 4) GCACTTCCCCTTGTAGCTGTCAAACTCGCATTTCGCGCCGTCGCGGGGGC 3'

2) Two degenerate primers to amplify part of the sequence encoding the peptide by PCR (oligos 4643 and 4644) .

aa5-aal9 : G K C Y K K D N I C K Y K A Q

GGAAAATGCTACAAAAAAGACAACATATGCAAATACAAAGCACAA (SEQ ID No. 5) C G T T G G T T C T G T G C G G T G

T T

Linkl-TACAAAAAAGACAACAT 3' (SEQ ID No. 6) PCR primer T G G T T 32 17mer

+18mer linkl

Oligo 4643 aa33-a49

C P R D G A K C E L D S Y K G K C TGCCCACGAGACGGAGCAAAATGCGAACTAGACTCATACAAAGGAAAATGC T C C T C C G T G C T C T G C G T G G G G G G G

T T T T T T T

AGA TTA AGC G G T

3'CGATTCACACTCGAACT - Link2 PCR primer C T G T 32 G 17mer T +18mer link2

Oligo 4644 (SEQ ID Nos. 7 and 8)

Linkl= CTGCAGGTCGACGGATCC 3' (SEQ ID No. 9)

Link2= GTGAATTCCCGGGGATCC 3' (SEQ ID No. 10)

It was decided to isolate a genomic clone rather than a cDNA clone, because the published cDNA sequence could be expected to be less than full length, and the peptide could be part of a large primary translation product.

The long oligonucleotide probes (4641 and 4642) were used for a Southern hybridization (2xSSC + 0.1% SDS, 50°C) with genom¬ ic DNA isolated from the above mentioned A. giganteuε strain (the primers were kinated with 7~³²P-ATP) . With oligo 4641 a single hybridizing band was seen for a variety of digestions of the genomic DNA.

The PCR primers (4643 and 4644) amplified a 164 bp fragment which could easily be aligned to the published sequence, under the assumption of the presence of a 57 bp intron sequ- ence. This fragment was considered to be an ideal probe for screening a library. The PCR was carried out as described above with the exception that 40 μmole of each primer were used and that 25 cycles with the annealing temperature at 45°C were conducted using genomic DNA as a template.

However, attempts of making an A . giganteus library in pUC failed repeatedly, and in an alternative attempt new outward primers (oligos 4708 (27mer, SEQ ID No. 11) and 4709 (26 mer, SEQ ID No. 12)) were synthesized on the basis of the cloned PCR fragment obtained with primers 4643 and 4644 as described above.

These oligonucleotide probes were used to clone the rest of the peptide encoding sequence and adjacent sequences as shown in fig. 1. Thus, genomic A . giganteuε DNA was cut with Bgl II, relegated and PCR'ed (according to the standard PCR protocol (Lewis and Gelfand, 1990) , with the exception that 30 cycles were performed and that the annealing temperature was 55°C) with primers 4708 and 4709. An approx 1300 bp fragment was amplified (as expected from the genomic Sout¬ hern) and cloned into a pCRII T/A vector (Invitrogen Corpora- tion) . Dideoxy sequencing of the insert confirmed that the amplified fragment contained the remaining part of the pepti¬ de encoding gene and adjacent sequences. The resulting DNA sequence is shown in SEQ ID No. 1.

A dot matrix of the sequence shown in SEQ ID No. 1 against the published cDNA sequence shows the presence of two in- trons. These encompass nucleotides 145-234 and 316-372 (when using the numbering obtained when the A of the initiating methionine codon is set to be nucleotide 1) .

The sequence shown in SEQ ID No. 1 contains one frameshift relative to the published cDNA sequence, namely in position 124 (using the numbering defined above) .

By analysis of the nucleotide sequence shown in SEQ ID No. 1 and encoding the antifungal polypeptide it is found that the sequences encodes a 43 amino acid long prepropeptide prece¬ ding the mature antifungal peptide (nucleotides 1-129) (pro¬ vided that there is no in frame, open frame 3rd intron) . The nucleotide sequence shown in SEQ ID No. 1 is in complete agreement with the amino acid sequence of the antifungal polypeptide shown in SEQ ID No. 2 (determined as described in Example 1) Thus the sequence contains the five amino acid substitutions relative to published sequence specifically mentioned in Example 1 above.

EXAMPLE 3

Expression of an antifungal A . giQanteuε polypeptide

To express the antifungal peptide in Aεpergilluε oryzae , the expression plasmid pMT1597 was constructed as follows:

Primers 4899 (SEQ ID No. 13) and 4826 (SEQ ID No. 14) were used to PCR amplify the entire gene encoding the antifungal peptide using A . giganteuε genomic DNA as a template (anne¬ aling at 50°C, 30 cycles) and at the same time placing BamHI sites just 5' and 3' to the coding sequences. THe PCR product was cut with BamHI and ligated into the BamHi-cut and dephos- phorylated vector pMHan 37 obtained as follows:

The p960 plasmid, described in EP 305,216 and used for ex¬ pression of Humicola lanuginoεa lipase, was modified by replacing 60 basepairs of the 5' untranslated region of the Aεpergilluε oryzae TAKA promotor just upstream to the Humico- la lanuginoεa lipase encoding gene with the corresponding 5' untranslated region from the Aεpergilluε nidulanε TPI (trio- sephosphate isomerase) gene. A synthetic oligonucleotide containing the 5' untranslated region from A . nidulanε TPI (triosephosphate isomerase) gene, flanked at each end by 20 bases homologous to p960 sequences just outside the untrans¬ lated region, was used in a PCR reaction together with anot¬ her primer covering the BssHII site in the TAKA promotor region. As the mutagenization primer covers the BamHI site close to the ATG start codon, the PCR fragment was digested with BamHI and BssHII, and recloned into p960 digested with BssHII and partially with BamHI, to give the above pMHan37 plasmid.

One clone (pMT1597) with the antifungal peptide encoding gene inserted in the correct orientation under control of the A. oryzae TAKA amylase promoter (described in EP 238 023) was identified. The sequence of the PCR amplified fragment was verified to be identical to the one contained within SEQ ID No. 1.

pMT1597 was cotransformed into A.oryzae A1560 with the selec¬ tive plasmids ToC90 carrying the amdS from A . nidulanε using the procedure described in WO 91/17243. The transformants were selected on Cove minimal plates with glucose and aceta- mide as carbon and nitrogen source, respectively. Transfor¬ mants were reisolated twice and grown in 10ml of YP (1% yeast extract, 2% peptone) + 2% maltose.

The expression levels for the pMT1597 transformants were analyzed by rocket immuno electroforesis using highly purifi- ed native antifungal peptide as standard. 12 out of 16 trans¬ formants produced A . giganteuε peptide at concentrations between 10 and 100 mg/1.

The presence of the expression plasmid was later confirmed by Southerns of at least some transformants. On this basis, two pMT1597 transformants were chosen, namely the highest pro¬ ducer (MT1600) and one producing only 25 % of the best (MT1601) .

MT1600 and 1601 were then cultivated in tanks. Glycerol was used for inoculum and start of the fermentation, while the feed contained maltose. The best tank gave more than 200 mg/1 of the antifungal peptide of the invention. The peptide was purified and shown to be identical to the peptide obtained from A . giganteuε as described in Example 1. Purification of recombinant antifungal peptide The recombinant peptide was purified as described in Materi¬ als and Methods with the modifications mentioned below.

The cation exchange column was equilibrated with 20 mM sodium phosphate pH 7.5 and pH in the centrifugated fermentation broth was adjusted to 7.5 before it was applied to the resin. The antifungal peptide was eluted with 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.5.

The purity of the purified recombinant peptide was higher than 95% judged by HPLC analyses as described in example 1. By amino acid sequencing as described in Materials and Met¬ hods, the purified peptide was found to have the same amino acid sequence as the native peptide. The purified peptide was diluted with Milli Q water to a concentration of 1.0 mg/ml based on the absorbance at 280 nm and used in the in vivo assay described in example 5.

EXAMPLE 4

Analysis of antifungal activity in vitro

A number of bacteria and fungi, including plant pathogens, was tested for sensitivity towards the antifungal protein of the invention isolated from the A . giganteuε strain A3274.

Strains of Aεpergilluε oryzae , Aεpergilluε niger, Bacilluε εubtilliε , Pεeudomonaε aeroginoεa and Saccharomyceε cerevicieae were tested using the in vitro assay I described below.

The plant pathogens, Botrytiε cinerea , Fuεarium oxysporium and Rhizoctonia εolani were tested using the in vitro assay I described below.

The petridishes used for assaying each of the bacteria and fungi were made from agar prepared as follows: For Aεpergilluε oryzae (26°C) , Aεpergilluε niger (26°C) , Botrytiε cinerea (26°C) : 2-6 ml of a suspension prepared from an agar slant culture containing 10⁶ spores per ml were mixed with 100 ml of YPG-1-agar (yeast extract (0.4%), KH₂P0₄ (0.1%), MgS0₄, 7H₂0 (0.05%), glucose (1.5%), Agar (1.5%) (48°C) .

Bacilluε subtiliε (30°C) , Pseudomonas aeroginosa (37°C) and Saccharomyceε cerevicieae (26°C) : A suspension prepared from an agar slant culture was suitably diluted so that a mixture of 6 ml of the diluted suspension and 100 ml of an Antibiotic medium-2 (Difco) (48°C) contained about 10⁶ spores per ml.

Fuεarium o ysporium (26°C) : A 8 days old PDA slant culture containing the sporulated fungus was washed with 10 ml of H₂0 and Tween. The spore suspension was filtered through a glass filter (G-1) . 6 ml of the filtrate were mixed with 100 ml of PD-agar (48°C) .

Rhizoctonia εolani (26°C) : The mycelium from a 14 days old slant culture (PDA) was washed with 10 ml of H₂0 and Tween. The resulting mycelium suspension was inoculated into an Erlenmeyer flask containing 100 ml of YPG-1. The flask were incubated for 6 days at 26°C under shaking (200 rpm) . The culture broth was homogenized for 1 minute and 6 ml of the resulting homogenate were mixed with 100 ml of PDA (48°C) .

Bioassay in vitro - I The assay was carried out in petridishes (14 cm) , each pre¬ pared from 35 ml of an agar suspension prepared as described above. In 4 mm holes made in the agar, 15 μl of the purified protein solution, the fermented broth and the sterile fil¬ trate obtained as described above were applied. Plates con- taining bacteria were incubated for one day at the tempera¬ tures indicated in the list of bacteria. Plates containing fungi were incubated for two days at 26°C. Results

Antifungal protein preparation

The numbers indicate mm of inhibition zone.

A is a sample of the fermented broth of A3274 obtained as described above.

B is a sample of a sterile filtrate of the fermented broth obtained as described above.

C is a sample of the purified antifungal protein of A3274 present in the buffer described in Example 1. The concentra¬ tion of the antifungal protein was 1 mg/ml.

D and E are buffer solutions used for control,

Bioassay in vitro - II for plant pathogens

The fungi were grown in petridishes containing PD-agar for 8 days at 26°C. Three pieces of agar, each of 1 cm², were cut from the mycelium and added to an Erlenmeyer flask containing 100 ml of PD bouillon. The flask was incubated on a shake board at 140 rpm for 6 days at 18-19°C. After centrifugation, the biomass and 30 ml of the supernatant were homogenized for about 1 minute. 10 ml of the resulting homogenized suspension were mixed with 100 ml of PD-agar (48°C) . 35 ml of the agar suspension were poured into 14 cm Petridishes and allowed to solidify, after which 4 mm holes were punched in the agar. 15 or 30 μl of the purified protein solution were applied to each hole. The plates were incubated for two days at 26°C.

Ptrenophora tereε

Sclerotinia εchlerotium Phoma lingam Monilinia fuctigena Helminthoεporia sativuε

The antifungal protein of the invention did not have any inhibitory effect on Aspergillus oryzae.

As it can be seen from the above results, the antifungal protein of the invention show interesting fungicidal activity against important plant pathogens of barley (P. tereε) , cabbage (P. lingam) and cereals (C. εativuε) .

EXAMPLE 5

Bioassay in vivo

Effect of antifungal polypeptideε againεt powdery mildew (Eryεiphe graminiε f . εp. hordeii) infecting barley plantε 8-10 days old barley plants of the cultivar "Canor", grown under green-house conditions (5 plants per pot, 5 cm pots) , were sprayed with 0.3 ml of a solution containing 1 mg/ml of the antifungal protein obtained as described in Example 1 above (from A3274) dissolved in lOmM Phosphate buffer, pH 7-8 using a handhold "Aerograph Super 63" sprayer. A solution containing no active ingredient was used as control. The plants were dried for 2-3 hours before they were inoculated with conidia from eight to ten days old colonies of E. gram- iniε grown on barley plants of the same cultivar. Inoculation was done by gently shaking the infected plants above the sprayed plants. The inoculum density on the sprayed plants was in from 10 to 30 conidia per mm².

In order to provide the fungus with optimal conditions the plants were incubated in a plant growth room (16 hours light; 10-12.000 lux, 8 hours darkness at 18-20°C for eight days and subsequently scored for E. graminiε infection.

No phytotoxicity was observed from spraying the active ingredient on the plants.

1 mg/ml 1 % of the leaf area covered by the fungus

Buffer alone 50 % of the leaf area covered by the fungus

Effect of antifungal polypeptides against grey mold (Botrytiε cinerea) infecting tomato plants

10-14 days old tomato plants of the cultivar "First In Field" grown under green-house conditions (1 plant per pot, 5 cm pots) were sprayed with 0.3 ml of a solution containing 1 mg/ml of the antifungal protein obtained as described in Example 1 above (from A3274) and 1 mg/ml of the recombinant antifungal peptide described in Example 3 above, respective¬ ly, dissolved in 10 mM Phosphate buffer, pH 7-8 using a handhold "Aerograph Super 63" sprayer. A solution containing no active ingredient was used as control. The plants were dried for 2-3 hours before they were inoculated with a spore suspension containing 5 x 10⁵ spores of B . cinerea per ml in 25 % (v/v) autoclaved grape fruit juice. The inoculation was carried out with a hand held sprayer ("Wagner W 50" type 0237) . In order to provide the fungus with optimal conditions the plants were incubated at high humidity in the darkness at 19°C for five days and subsequently scored for the level of B . cinerea infection. No difference in antifungal activity of the purified native antifungal polypeptide and the recombi¬ nant antifungal polypeptide could be observed.

No phytotoxicity was observed from spraying the active ingredient on the plants.

1 mg/ml^* 90 % protection (100 % protection equals no attack from the pathogen) .

Buffer alone 10-20 % protection.

of native as well as recombinant antifungal polypeptide.

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5 Nakaya, K. et al., Eur. J. Biochem. 193, 31-38, 1990, "Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus";

Olson, B.H. et al.. Applied Microbiology, May, 1965, Vol. 13, No. 3.: 314-321, "Alpha Sarcin, a New Antitumor Agent";

0 Protein Engineering, Editors Oxender, D.L. and Fox, C.F., Alan R. Liss, Inc. New York (1987)

Russell et al., Nature 304. 1983, pp. 652-654

Sacco et al., J. Biol. Chem. 258, 5811-18, 1983; R.K. Saiki et al. (1988), Science 239. 1988, pp. 487-491;

Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989;

Wnendt, S. et al., Nucleic Acids Research, Vol. 18, No. 13: 5 3987, 1990, "Cloning and nucleotide sequence of a cDNA encoding the antifungal-protein of Aspergillus giganteus and preliminary characterization of the native gene".

Yelton et al., Proc. Natl. Acad. Sci. USA 81, 1984, pp. 1470- 1474

0 Young et al., Plenum Press, New York, 1982)

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(E) COUNTRY: DENMARK

(F) POSTAL CODE (ZIP) : DK-2880

(G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 4449 3256

(I) TELEX: 37304

(ii) TITLE OF INVENTION: Antifungal peptide from Aspergillus gigan¬ teus

(iii) NUMBER OF SEQUENCES: 14

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) ∞MPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 831 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus giganteus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

OGCCTGCAGG TGAGGATGTG CTCCTGGTCA TTCAGGCAAT GAGATATCCA CGTOGAGAGA 60 GATGACATTC TCGAGCTGGG AGATTCCATC CAGTOGTAGC ATCAGCATCT CTCOGTITTG 120

GAGAGAAAAT GAGCΑTCGCT GGTCTATAAA AGGGTGCCAT CAACCCAGGG TTTTTTGGAA 180

AGTATCAATA TCAACAACCA CAAAGAAACA CTCTGATCTA TCCAATCCAC TAOGCAAAAA 240 GCAAATAAAA CCCCrø-rCAC CCCCTATCTC AATTCATCAT GAACTTCGTT TCTCTTGCTT 300

CTTTGGGATT CGCCCTCGTT GCTGCCCTTG GCC^CGGTTGC C-ΑCCCCCATT GAAGCCGATT 360

CTCTCACCGC TGGTGGTCTG GATGCAAGAG AOGAGAGCGC AGTΓΓTGGCC ACAIACCCCG 420

GCGTAGGTCT TCTCTTCTGA AAGCCCCAAA CCCAGCCAGT GTCCGAGOGC GOIATGGGTC 480 TTAACIAACC TCCG ITI'IC COXCTCCTT AGAAATGCTA CAAGAAGGAT AATATCTGCA 540

ACTACAAGGC ACAGAGTGGC AAGACTGGTA TTTGCAAGTG CTATGTGAAA AGGGTATGTA 600

CTICTGCACT GGCGGCOGGG AGACTAGAGT GAAGTAGCTG ATGTTCTTAG TGCCCCCGGG 660

ACGGTGCGAA ATGCGAGCTT GACAGCTACA AGGGGAAGTG CCΑCTGCTAG AOGCTGAGCA 720

AAGGGACGAA GCAGGGTGGG GATATITTAT TCTGCTCTGC GGATCATACG AGTCATTCIT 780 TACTGTAAAG AGACGTGGGG AGGGTATTCA ACATACCGAC TCATGATCTA G 831

(2) INPORMATTCN FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOI-ECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTT-SENSE: NO (v) - AGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus giganteus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Ala Thr Tyr Pro Gly Lys Cys Tyr Lys Lys Asp Asn lie Cys Lys Tyr 1 5 10 15

Lys Ala Gin Ser Gly Lys Thr Gly lie Cys Lys Cys Tyr Val Lys Arg 20 25 30

Cys Pro Arg Asp Gly Ala Lys Cys Glu Leu Asp Ser Tyr Lys Gly Lys 35 40 45 Cys His Cys 50 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTEIRISTICS:

(A) LENGTH: 57 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic)

(iii) HYPOTHETICAL: NO

(iii) ANTT-SENSE: NO (v) ERAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GGCAAATGCT ACAAGAAGGA TAATATCTGC AAGTACAAGG CACAGAGOGG CAAGACT 57

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MO]-xECULE TYPE: DNA (synthetic) (iii) HYPCTHETICAL: NO (iii) ANTT-SENSE: YES

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCACTTCCCC TTGTAGCTGT CA CTCGCA TTTCGCGCCG TCGCGGGGGC 50

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MO---E>CUI-E TYPE: cDNA

(iii) HYPOTHETICAL: YES (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGNAARTGYT AYAARAARGA YAAYATHTGY AARTAYAARG CNCAR 45

(2) INEORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOI-ECULE TYPE: DNA (synthetic)

(iii) HYPCTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FIG ENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TAYAARAARG AYAAYAT 17

(2) TNPOR ATION FOR SEQ ID NO: ! :■

(i) SEQUENCE CBARACTE-3ISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TGYCCNCGNG AYGGNGCNAA RTGYGARCTN GAYTCNTAYA ARGGNAARIG Y 51

(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MO--_ECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TCAAGYTCRC AYTTNGC 17

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOI__CULE TYPE: DNA (synthetic) (iii) HYPCTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTGCAGGTCG ACGGATCC 18

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) M0---ECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GTGAATTCCC GGGGATCC 18

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) M0---ECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTAGAGTGAA GTAGCTGATG TTCTTAG 27

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE (CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: YES (v) FΪ^GMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTITTCACAT AGCACTTGCA AATACC 26

(2) -CNFOR ATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) M0I-EΪCULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TOGGATCCTC -ATTCATCAT G GTTCG 28

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) M0---ECULE TYPE: DNA (synthetic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: YES

(v) FRAG ENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CGGGATCCCC ACCCTGCITC GTCC 24

Claims (41)

1. A DNA construct comprising a DNA sequence encoding an antifungal polypeptide, which sequence comprises the nucleo¬ tide sequence shown in SEQ ID No. 1 or an analogue thereof, which

i) hybridizes with a DNA sequence comprising the nucleoti¬ de sequence shown in SEQ ID No. 1, or with a probe hybridizing with the nucleotide sequence SEQ ID No. 1,

ii) encodes a polypeptide which reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequence shown in SEQ ID No. 2, and/or

iii) encodes a polypeptide having the amino acid sequence shown in the appended SEQ ID No. 2 or a sequence homo- logous thereto,

provided that the DNA sequence is different from one which encodes the polypeptide having the amino acid sequence shown in SEQ ID No. 2 except for an asparagine in position 4, an alanine in position 24, a lysine in position 32, a phenylala- nine in position 42 and a tyrosine in position 50.

2. A DNA construct according to claim 1, which encodes a polypeptide which comprises the amino acid sequence shown in SEQ ID No. 2, in which the Pro in position 4 has been repla¬ ced with an Asp, the Gly in position 24 has been replaced with an Ala, the Arg in position 32 has been replaced with a Val, the Leu in position 42 has been replaced with a Phe and/or the His in position 50 has been replaced with a Tyr.

3. A DNA construct according to claim 1 or 2, wherein the DNA sequence is derived from a mammal, an insect, a plant, a protozoae, an algae, or a microorganism.

4. A DNA construct according to claim 3, wherein the microor¬ ganism is a bacterium or a fungus.

5. A DNA construct according to claim 4, wherein the DNA sequence is derived from a fungus of the genus Aεpergilluε ,

5 in particular of the species A . giganteuε , A. pallidus , A . clavatuε , A. longiveεica , A . rhizopoduε and A . clavatona¬ nicuε .

6. A DNA construct according to claim 5, wherein the DNA sequence is derived from the A . giganteuε strain CBS 526.65.

107. A DNA construct according to any of the preceding claims in which the DNA sequence is a cDNA sequence, a genomic DNA sequence or a synthetic DNA sequence or a mixed cDNA, genomic and/or synthetic DNA sequence.

8. An antifungal polypeptide comprising the amino acid

15 sequence shown in SEQ ID NO. 2, or a variant thereof, which

1) reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequ¬ ence shown in SEQ ID NO. 2, or

2) has an amino acid sequence which is substantially

20 homologous with the amino acid sequence of the polypep¬ tide shown in SEQ ID NO. 2,

provided that the variant is different from the polypeptide having the amino acid sequence shown in SEQ ID No. 2 except for an asparagine in position 4, an alanine in position 24, a 25 lysine in position 32, a phenylalanine in position 42 and a tyrosine in position 50.

9. A polypeptide according to claim 8, which comprises the amino acid sequence shown in SEQ ID No. 2, in which the Pro in position 4 has been replaced with an Asp, the Gly in posi-

30 ton 24 has been replaced with an Ala, the Arg in position 32 has been replaced with a Val, the Leu in position 42 has been replaced with a Phe and/or the His in position 50 has been replaced with a Tyr.

10. The polypeptide according to claim 8 or 9, which is

5 obtainable from a mammal, an insect, a plant, a protozoae, an algae, or a microorganism.

11. The polypeptide according to claim 10, wherein the micro¬ organism is a bacterium or a fungus.

12. The polypeptide according to claim 11, which is derived 10 from a fungus of the genus Aεpergilluε , in particular of the species A . giganteuε , A . pallidus , A . clavatuε , A . longiveεi- ca , A. rhizopoduε and A . clavatonanicuε .

13. The polypeptide according to claim 12, which is derived from the A. giganteus strain CBS 526.65.

1514. The polypeptide according to claim 8, which is encoded by a DNA sequence according to any of claims 1-7.

15. A recombinant expression vector comprising a DNA con¬ struct according to any of claims 1-7.

16. A cell comprising a DNA construct according to any of 20 claims 1-7 or a vector according to claim 15.

17. The cell according to claim 16, which is a microbial cell.

18. The cell according to claim 17 which is a bacterial cell or a fungal cell.

2519. The cell according to claim 18, in which the bacterial cell is a cell of a gram-positive bacterium such as Bacilluε or Streptomyceε or a cell of a gram-negative bacterium such as Eεcherichia , the fungal cell is a yeast cell such as a cell of Saccharomyces or a cell of a filamentous fungus such as Aεpergillu .

20. The cell according to claim 18, which is a cell of the fungal species A . oryzae .

21. A method of producing an antifungal polypeptide compri¬ sing the amino acid sequence shown in SEQ ID NO. 2, or a variant thereof, which

1) reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequ- ence shown in SEQ ID NO. 2, or

2) has an amino acid sequence which is substantially homologous with the amino acid sequence of the polypep¬ tide shown in SEQ ID NO. 2,

which method comprises

(a) inserting a DNA construct encoding the polypeptide into a suitable expression vector,

(b) transforming a suitable host cell with the recombinant expression vector of step (a) ,

(c) culturing the transformed host cell in a suitable cul- ture medium under conditions conducive to the produc¬ tion of the polypeptide, and

(d) recovering the polypeptide from the host cell or cul¬ ture medium obtained in step (c) .

22. The method according to claim 21, in which the DNA con- struct is the DNA construct defined in any of claims 1-7, the expression vector is the vector defined in claim 15, and/or the host cell is as defined in any of claims 16-20.

23. The method according to claim 21, in which the DNA con¬ struct comprises a DNA sequence encoding an antifungal poly¬ peptide having the amino acid sequence shown in SEQ ID No. 2 except for an asparagine in position 4, an alanine in posi- tion 24, a lysine in position 32, a phenylalanine in position 42 and a tyrosine in position 50, and the host cell is a a bacterial cell or a cell of a filamentous fungus.

24. The method according to any of claims 21-23, which fur¬ ther comprises

(e) modifying the polypeptide or variant obtained in step (d).

25. A method of producing an antifungal polypeptide as defi¬ ned in any of claims 8-14, comprising cultivating a micro¬ organism which, in nature, is capable of producing the poly- peptide on a suitable culture medium and under conditions allowing the production of the polypeptide, and recovering the polypeptide from the resulting biomass and/or fermented culture medium.

26. The method according to claim 25, in which the microorga- nism is a fungus, in particular of the genus Aεpergilluε , and more particularly of the species A . giganteuε , A . palliduε , A. clavatuε , A. longiveεica , A . rhizopodus and A. clavatona¬ nicuε.

27. An antifungal polypeptide produced by the method accor- ding to any of claims 21-26.

28. A fungicidal composition comprising, as an active ingredient, an antifungal polypeptide according to any of claims 8-14 or 26.

29. The fungicidal composition according to claim 27, which further comprises an additional biocidal agent and/or a plant growth regulator.

30. The fungicidal composition according to claim 28 or 29, comprising a fermented culture medium containing the anti¬ fungal polypeptide and obtained by the method according to any of claims 21-26.

5 31. A method of producing a fungicidally active composition, comprising mixing an antifungal polypeptide as defined in any of claims 8-14 and 27 with an inert carrier.

32. The method according to claim 31, in which an additional biocidally active compound and/or a plant growth regulator 0 are/is mixed with the antifungal polypeptide and the inert carrier.

33. A method of combating or controlling fungi at a locus infested or liable to be infested with a fungus, comprising applying to said locus an antifungal polypeptide as defined 5 in any of claims 8-14 and 27, a fermentation medium contai¬ ning an antifungal polypeptide and obtained by the method according to any of claims 21-26 or a fungicidal composition according to any of claims 27-29.

34. The method according to claim 33, in which the locus is a 0 part of a plant.

35. The method according to claim 33 or 34, wherein the fungus to be controlled or combated belongs to the class Aεcomyceteε , Deuteromyceteε , or Oomyceteε .

36. The method according to claim 35, wherein the fungus

25 belongs to the genus Pyrenophora , especially the species P. tereε , the genus Phoma , especially the species P. lingam , the genus Cochlioboluε, especially the species C. εativuε .

37. Use of an antifungal polypeptide as defined in any of claims 8-14 for controlling or combatting fungi.

30 38. The use according to claim 37, in which the fungus to be controlled or combated belongs to the class Aεcomyceteε , Deuteromycetes , or Oomycetes .

39. The use according to claim 38, wherein the fungus belongs to the genus Pyrenophora , especially the species P. teres , the genus Phoma , especially the species P. lingam , the genus Cochlioboluε , especially the species C. εativuε .

40. An antifungal polypeptide according to any of claims 8-14 or 26 for use as an antifungal veterinarian or human drug.

41. Use of an antifungal polypeptide according to any of claims 8-14 or 27 for use in the preparation of a veterinari- an or human drug for the treatment of a fungal infection.