HK1110336A - Recombinant expression of defensins in filamentous fungi - Google Patents

Description

Recombinant expression of defensins in filamentous fungi

Technical Field

The present invention relates to recombinant expression of defensin antimicrobial peptides in filamentous fungi.

Background

Defensins belong to a class of small antimicrobial peptides. They are capable of killing a broad spectrum of microorganisms, some of which are becoming increasingly resistant to traditional antibiotics. For this reason, there is increasing interest in being able to produce defensins in large quantities at low cost.

Because defensins typically contain only 30-50 amino acid residues, they are often difficult to produce efficiently by using recombinant fermentation methods. Chemical peptide synthesis is an alternative method, but it is too expensive when the peptide exceeds 25-30 amino acid residues. Another complication is that defensins contain special cysteine patterns (cysteine patterns) which are difficult to produce by chemical synthesis.

It is therefore an object of the present invention to provide a method for obtaining improved expression levels of defensin antimicrobial peptides in recombinant fermentation of filamentous fungi.

Summary of The Invention

The inventors of the present invention have found that by inserting one or more intron sequences into a nucleic acid construct that directs the expression of a defensin, the recombinant expression level can be improved by more than 50% compared to the case of using a nucleic acid construct without intron sequences. The intron sequence may be inserted anywhere in the nucleic acid construct, for example in the mature defensin coding sequence, or even in the signal peptide coding sequence.

Accordingly, the present invention relates to a recombinant filamentous fungal host cell comprising a nucleic acid construct comprising an exogenous nucleic acid sequence encoding a defensin and one or more intron sequences.

In a second aspect, the present invention relates to a method of recombinantly producing a defensin in a filamentous fungal host cell comprising culturing a filamentous fungal host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences; and recovering the defensin peptide.

In a third aspect, the present invention relates to the use of a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences for improving the recombinant expression level of a defensin in a filamentous fungal host cell.

Definition of

Antimicrobial activity: the term "antimicrobial activity" is defined herein as an activity that is capable of killing or inhibiting the growth of microbial cells. In the context of the present invention, the term "antimicrobial" means that there is a bactericidal (bacteriostatic) and/or bacteriostatic (bacteriostatic) and/or fungicidal (fungicidal) and/or fungistatic (fungistatic) effect and/or virucidal effect, wherein the term "bactericidal" is to be understood as being capable of killing bacterial cells. The term "bacteriostatic" is to be understood as a bacterial cell capable of inhibiting bacterial growth, i.e. inhibiting growth. The term "fungicidal" is to be understood as capable of killing fungal cells. The term "fungistatic" is to be understood as a fungal cell capable of inhibiting fungal growth, i.e. inhibiting growth. The term "virucidal" is understood to mean capable of inactivating a virus. The term "microbial cell" represents a bacterial or fungal cell (including yeast).

In the context of the present invention, the term "inhibiting the growth of microbial cells" means that the cells are in a non-growing state, i.e. they are not able to proliferate.

For the purposes of the present invention, the compounds can be prepared according to Lehrer et al, j.immunol.methods, 137 (2): 167-174(1991) was used to determine antimicrobial activity. Alternatively, antimicrobial activity can be determined according to the NCCLS guidelines of the CLSI (Clinical and Laboratory Standards Institute), previously known as the National Committee for Clinical and Laboratory Standards.

After 8 hours of incubation at 20 ℃ (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and especially after 30 minutes) in a 25% (w/w) aqueous solution, preferably in a 10% (w/w) aqueous solution, more preferably in a 5% (w/w) aqueous solution, even more preferably in a 1% (w/w) aqueous solution, most preferably in a 0.5% (w/w) aqueous solution, of a defensin with antimicrobial activity, the defensin with antimicrobial activity may be able to reduce the viable cell number of escherichia coli (DSM1576) to 1/100.

When added at a concentration of 1000ppm, preferably at a concentration of 500ppm, more preferably at a concentration of 250ppm, even more preferably at a concentration of 100ppm, most preferably at a concentration of 50ppm, and in particular when added at a concentration of 25ppm, a defensin with antimicrobial activity may also be able to inhibit the growth halo (outgrowth) of escherichia coli (DSM1576) in the growth substrate (growth) of a microorganism at 25 ℃ for 24 hours. A defensin with antimicrobial activity may be able to reduce the number of viable cells of Bacillus subtilis (ATCC6633) to 1/100 after 8 hours of incubation at 20 ℃ (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and especially after 30 minutes) in a 25% (w/w) aqueous solution, preferably in a 10% (w/w) aqueous solution, more preferably in a 5% (w/w) aqueous solution, even more preferably in a 1% (w/w) aqueous solution, most preferably in a 0.5% (w/w) aqueous solution, and especially in a 0.1% (w/w) aqueous solution of the defensin with antimicrobial activity.

When added at a concentration of 1000ppm, preferably when added at a concentration of 500ppm, more preferably when added at a concentration of 250ppm, even more preferably when added at a concentration of 100ppm, most preferably when added at a concentration of 50ppm, and particularly when added at a concentration of 25ppm, a defensin having antimicrobial activity may also be capable of inhibiting the growth halo of bacillus subtilis (ATCC6633) in a growth substrate for a microorganism for 24 hours at 25 ℃.

The defensins of the invention have the amino acid sequence represented by SEQ ID NO: 2, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the antimicrobial activity of a defensin consisting of the amino acid sequence shown as amino acids 1 to 42.

cDNA: the term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are normally present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor of mRNA that is processed through a series of steps and then visualized as mature spliced mRNA. These steps include the removal of intron sequences by a process known as splicing. Thus, cDNA derived from mRNA lacks any intron sequences.

Nucleic acid construct: the term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that is modified to contain nucleic acid fragments in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

And (3) control sequence: the term "control sequences" is defined herein to include all components which are necessary or advantageous for expression of a polynucleotide encoding a defensin. For nucleotide sequences encoding defensins, each control sequence may be native or foreign. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers (linkers) for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a defensin.

Operatively connected to: the term "operably linked" herein denotes a structure (restriction) in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a defensin.

A coding sequence: as used herein, the term "coding sequence" means a nucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG. The coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.

Expressing: the term "expression" includes any step involved in defensin production, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: the term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide encoding a defensin peptide operably linked to additional nucleotides that provide for its expression.

Host cell: the term "host cell" as used herein includes any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct of the present invention.

Modification: the term "modification" refers herein to any chemical modification of a defensin. Modifications may be substitutions (substitutions), deletions and/or insertions of amino acids, and substitutions (displacements) of amino acid side chains; or the use of unnatural amino acids within amino acid sequences that have similar properties. The modification may specifically be amidation (amidation), for example, C-terminal amidation.

Identity: the relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For the purposes of the present invention, the degree of identity between two amino acid sequences is determined by using the program FASTA, which is included in the FASTA package, version 2.0 × (see W.R. Pearson and D.J.Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85: 2444-. The scoring matrix used is BLOSUM50, the gap (gap) penalty is-12, and the gap extension penalty is-2.

The degree of identity between two nucleotide sequences is determined using the same algorithms and software packages as described above. The scoring matrix used is an identity matrix, the gap penalty is-16, and the gap extension penalty is-4.

Alternatively, the alignment of the two amino acid sequences was determined by using the Needle program from EMBOSS software package (http:// EMBOSS. org) version 2.8.0. The Needle program performs the global alignment algorithm described in Needleman s.b. and Wunsch c.d. (1970), j.mol.biol.48: 443-. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the invention ("the sequence of the invention", e.g., amino acids 1 to 40 of SEQ ID NO: 2) and a different amino acid sequence ("the foreign sequence") is calculated as the number of exact matches in the overlapping region of the alignment of the two sequences divided by the shortest of the length of the "sequence of the invention" or the length of the "foreign sequence". Results are expressed as percent identity.

An exact match occurs when the "sequence of the invention" and the "foreign sequence" have identical amino acid residues in the same position in the overlapping region. The length of a sequence is the number of amino acid residues in the sequence (e.g., amino acids 1 to 40 of SEQ ID NO: 2 are 40 in length).

Detailed Description

Defensin

The defensins of the invention are any antimicrobial peptide recognized by those skilled in the art as belonging to the class of defensins of antimicrobial peptides. To determine whether an antimicrobial peptide is a defensin of the invention, the amino acid sequence is preferably compared to the hidden Markov model profile (HMM profile) of the well known PFAM database (see example 6).

The defensins can be alpha-defensins, beta-defensins, theta-defensins, arthropod defensins, insect defensins, plant defensins.

The defensins may also be synthetic defensins which share unique characteristics of any class of defensins.

In an embodiment, the amino acid sequence of the defensin according to the invention comprises 4, 5, 6, 7, 8, 9 or 10 cysteine residues, preferably 6, 7, 8, 9 or 10 cysteine residues, more preferably 6, 8 or 10 cysteine residues, and most preferably 6 or 8 cysteine residues.

Examples of defensins include, but are not limited to, the alpha-defensins HNP-1 (human neutrophil peptide), HNP-2 and HNP-3; beta-defensin-12, Drosomycins defensins (Drosomycins), Helioticins (Heliomicin), gamma 1-purothionins (purothionins), insect defensin A and defensins disclosed in PCT application WO9953053 (RONE POULENC AGROCHIMIE) (10/21 1999) and WO02085934(ENTOMED) (10/31 2002), which are incorporated herein by reference; or SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14 and SEQ ID NO: 16 or an amino acid sequence having at least 60%, preferably 70%, more preferably 80%, even more preferably 90% and most preferably 95% identity to these sequences. The defensins of the invention may further comprise one or more chemical modifications compared to these amino acid sequences.

Alpha-defensins can be defined as antimicrobial peptides comprising the amino acid sequence:

C-X₁-C-X₂-C-X₃-C-X₄-C-C

-wherein:

X₁represents 1 amino acid; preferably, X₁Y, F, A, R, I, S, T, H or V; more preferably, X₁Y, F, A or R; even more preferably, X₁Y or F; most preferably, X₁＝Y；

X₂Represents 4 or 5 amino acids; preferably, X₂Represents 4 amino acids; more preferably, X₂＝Z₁-Z₂-Z₃-Z₄Wherein:

Z₁represents any amino acid; preferably, Z₁R, T or K; more preferably, Z₁＝R；

Z₂Represents any amino acid; preferably, Z₂＝R、I、T、K；

Z₃Represents any amino acid; preferably, Z₃R, P or G;

Z₄represents any amino acid; preferably, Z₄G, A or R;

X₃represents 9 amino acids; preferably, X₃＝Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-G-Z₈Wherein:

Z₁represents any amino acid; preferably, Z₁K, L or R;

Z₂represents any amino acid; preferably, Z₂R, F, A, S or G;

Z₃represents any amino acid; preferably, Z₃R, G, P or T; more preferably, Z₃R or G;

Z₄represents any amino acid; z₄E or Y; preferably Z₄＝E；

Z₅Represents any amino acid; preferably, Z₅R, H or S;

Z₆represents any amino acid; preferably, Z₆R, M or L;

Z₇represents any amino acid; preferably, Z₇N, S, Y or I;

Z₈represents any amino acid; preferably, Z₈T, S, Y or a;

X₄represents 9 amino acids; preferably, X₄＝Z₁-Z₂-Z3-Z₄-Z₅-Z6-Z₇-Z₈-Z₉Wherein:

Z₁represents any amino acid; preferably, Z₁R or I;

Z₂represents any amino acid; preferably, Z₂K, I, Y, F or L;

Z₃represents any amino acid; preferably, Z₃G, N, R or Q;

Z₄represents any amino acid; preferably, Z₄G, H or N;

Z₅represents any amino acid; preferably, Z₅R or L;

Z₆represents any amino acid; preferably, Z₆I, L, M, V or R;

Z₇represents any amino acid; preferably, Z₇Y, W, H or F;

Z₈represents any amino acid; preferably, Z₈T, R or a;

Z₉represents any amino acid; preferably, Z₉L, F or R; more preferably, Z₉L or F.

Beta-defensins can be defined as antimicrobial peptides comprising the amino acid sequence:

C-X₁-C-X₂-C-X₃-C-X₄-C-C

-wherein:

X₁represents 6 amino acids; preferably, X₁＝Z₁-Z₂-Z₃-Z₄-Z₅-Z₆Wherein:

Z₁represents any amino acid; preferably, Z₁R, V or L;

Z₂represents any amino acid; preferably, Z₂R, I, K or Q;

Z₃represents any amino acid; preferably, Z₃N or S;

Z₄represents any amino acid; preferably, Z₄G, K or R;

Z₅represents any amino acid; preferably, Z₅＝G；

Z₆Represents any amino acid; preferably, Z₆Q, I, V or F;

X₂represents 3 or 4 amino acids; preferably, X₂Represents 4 amino acids; more preferably, X₂＝Z₁-Z₂-Z₃-Z₄Wherein:

Z₁represents any amino acid; preferably, Z₁L, V, I, H or a;

Z₂represents any amino acid; preferably, Z₂P or Y;

Z₃represents any amino acid; preferably, Z₃S, I, N or G;

Z₄represents any amino acid; preferably, Z₄R, A or S;

X₃represents 9 amino acids; preferably, X₃＝Z₁-Z₂-Z₃-Z₄-Z₅-Z6-Z₇-Z₈-Z₉Wherein:

Z₁represents any amino acid; preferably, Z₁＝P；

Z₂Represents any amino acid; preferably, Z₂G, I, R or P;

Z₃represents any amino acid; preferably, Z₃Y, N, P, R, F or H;

Z₄represents any amino acid; preferably, Z₄T, M or Y;

Z₅represents any amino acid; preferably, Z₅R or K;

Z₆represents any amino acid; preferably, Z₆Q or I;

Z₇represents any amino acid; preferably, Z₇I or Q;

Z₈represents any amino acid; preferably, Z₈＝S；

Z₉Represents any amino acid; preferably, Z₉＝T；

X₄Represents 6 amino acids; preferably, X₄＝Z₁-Z₂-Z₃-Z₄-Z₅-Z₆Wherein:

Z₁represents any amino acid; preferably, Z₁Y, F, G or L;

Z₂represents any amino acid; preferably, Z₂G, H, P, L, R or T;

Z₃represents any amino acid; preferably, Z₃G, P or R;

Z₄represents any amino acid; preferably, Z₄K, P, R, G or Q;

Z₅represents any amino acid; preferably, Z₅V, A, I or G;

Z₆represents any amino acid; preferably, Z₆＝K。

Insect-defensins can be defined as antimicrobial peptides comprising the following amino acid sequence:

C-X₁-C-X₂-C-X₃-C-X₄-C-X₅-C

-wherein:

X₁represents 5-16 amino acids;

X₂represents 3 amino acids; preferably, X₂＝Z₁-Z₂-Z₃Wherein:

Z₁represents any amino acid; preferably, Z₁A or H;

Z₂represents any amino acid; preferably, Z₂A or R;

Z₃represents any amino acid; preferably, Z₃＝H；

X₃Represents 9-11 amino acids;

X₄represents 4 to 10 amino acids;

X₅represents 1 amino acid; preferably, X₅V, T, I, H, K, N or L.

In one embodiment, the defensins of the invention have more than one antimicrobial activity selected from the group consisting of antifungal activity, antibacterial activity and antiviral activity.

The defensins of the invention may be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in relation to a given source shall mean that the defensin encoded by the nucleotide sequence is produced by the source or by a strain into which the nucleotide sequence from the source has been inserted. In a preferred aspect, the defensin from a given source is secreted extracellularly.

The defensin of the invention may be a fungal defensin, and more preferably a yeast defensin, such as a Candida (Candida), kluyveromyces (kluyveromyces), Pichia (Pichia), Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), or Yarrowia (Yarrowia) defensin; or more preferably a filamentous fungal defensin such as Acremonium (Acremonium), Aspergillus (Aspergillus), Aureobasidium (Aureobasidium), Cryptococcus (Cryptococcus), Rhizopus (Filibasidium), Fusarium (Fusarium), Humicola (Humicola), pestis (Magnaporthe), Mucor (Mucor), Myceliophthora (Myiocephala), Ruminomyces (Neocallimastix), Neurospora (Neurospora), Paecilomyces (Paecilomyces), Penicillium (Penicillium), Ruminomyces (Piromyces), Schizophyllum (Schizophyllum), Talaromyces (Talaromyces), Thermoascus (Thermoascus), Thielavia (Thielavia), Tolypocladium (Tolypocladium), or Trichoderma (Trichoderma).

In a preferred aspect, the defensin is a saccharomyces carlsbergensis (s.carlsbergensis), saccharomyces cerevisiae (s.cerevisiae), saccharomyces diastaticus (s.diastaticus), dow garland (s.douglasii), kluyveromyces kluyveri (s.kluyveri), norbensis (s.norbensis), or oval yeast (s.oviformis) defensin with antimicrobial activity.

In another preferred aspect, the defensin is aspergillus aculeatus (a. aculeatus), aspergillus awamori (a. awamori), aspergillus fumigatus (a. fumigus), aspergillus foetidus (a. foetidus), aspergillus japonicus (a. japonica), aspergillus nidulans (a. nidulans), aspergillus niger (a. niger) or aspergillus oryzae (a. oryzae), fusarium culmornumerous (f. bactridioides), fusarium graminearum (f. cerealis), fusarium kukawakamura (f. crookllense), fusarium culmorum (f. culmorula), fusarium graminearum (f. graminearum), fusarium heterosporum (f. heterosporum), fusarium negundi (f. roseosporum), fusarium negundo, fusarium graminearum (f. oxysporum), fusarium oxysporum (f.sp.sp., fusarium sp.sp.sp.sp.sp., fusarium sp.sp.f.sp.sp.sp.sp.f.sp.sp.sp.sp.sp.sp.f.sp.sp.sp.f.sp.sp.sp.sp.sp.f.sp.sp.sp.sp.sp.sp.sp.f.f.sp.sp.sp.f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.f.sp.sp.sp.sp.sp.f.sp.sp.sp.f.sp.sp.sp.sp.sp.f.f.sp.sp.sp.sp.sp.f.f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.f.sp.sp.sp.f.f.sp.sp.sp.sp.sp.sp.sp, Penicillium purpurogenum (p. purpurogenum), trichoderma harzianum (t. harzianum), trichoderma koningii (t. konii), trichoderma longibrachiatum (t. longibrachiatum), trichoderma reesei (t. reesei), or trichoderma viride (t.viride) defensins.

It is to be understood that for the species mentioned above, the invention encompasses both the complete and incomplete stages as well as other categorical equivalents, e.g., anamorphs, regardless of their known species name. Those skilled in the art will readily recognize the identity of suitable equivalents.

Strains of these species are readily publicly available in a number of culture collections, for example, the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM, the fungal species Collection (CBS) and the Agricultural Research institute Patent Collection North Research Center (Agricultural Research Service Collection, Northern Research Center, NRRL).

In addition, these defensins may also be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) using the probes described above. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide can then be obtained by similarly screening a genomic or cDNA library of another microorganism. Once the polynucleotide sequence encoding the defensin is detected with the probe, the polynucleotide may be isolated or cloned using techniques well known to those of ordinary skill in the art (see, e.g., SAMBROOK,. Molecular Cloning: A Laboratory Manual.2.,. Cold Spring Harbor Laboratory Press, 1989.ISBN 0879693096).

Defensins of the invention also include fused defensins or cleavable fusion defensins wherein another defensin is fused to the N-terminus or C-terminus of the defensin or fragment thereof. A fused defensin is produced by fusing a nucleotide sequence (or portion thereof) encoding another defensin to a nucleotide sequence (or portion thereof) of the invention. Techniques for producing fusion defensins are known in the art and include ligating the coding sequences encoding the defensins so that they are in frame and expression of the fused defensin is under the control of the same promoter and terminator.

Nucleic acid sequence:

the invention also relates to polynucleotides having a nucleotide sequence encoding a defensin of the invention.

Examples of such polynucleotides include, but are not limited to, the polynucleotides disclosed in PCT application WO99/53053, the contents of which are incorporated herein by reference.

In a preferred embodiment, the nucleotide sequence is set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, in (b). In a further preferred embodiment, the nucleotide sequence is SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, the mature defensin coding region. The invention also encompasses nucleotide sequences encoding a polypeptide having the sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14 or SEQ ID NO: 16 or a mature defensin thereof, which due to the degeneracy of the genetic code differs from the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15.

techniques for isolating or cloning polynucleotides encoding defensins are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of a polynucleotide encoding a defensin of the invention from such genomic DNA can be accomplished by: antibody screening using, for example, the well-known Polymerase Chain Reaction (PCR) or expression libraries, to detect cloned DNA fragments with shared structural features. See, for example, Innis et al, 1990, PCR: a Guide to Methods and application, Academic Press, New York. Other nucleic acid amplification methods, such as Ligase Chain Reaction (LCR), Ligated Activated Transcription (LAT) and nucleotide sequence based amplification (NASBA), may also be used. The polynucleotide may be cloned from a strain of the genus Eurotium (Eurotium), Aspergillus (Aspergillus), Pseudoplectania (Pseudoplectania), Crassostrea (Crassostrea), Buthus (Mesobuthus) or other or related organisms and may thus be, for example, an allelic or species variant of the defensin coding region of the nucleotide sequence (speces variant).

The invention also relates to polynucleotides having a sequence identical to SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, having a nucleotide sequence with a degree of identity of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%, said polynucleotide encoding a defensin antimicrobial polypeptide.

Modification of the nucleotide sequence encoding a defensin of the invention may be necessary for the synthesis of a defensin substantially similar to the defensin. The term "substantially similar" to a defensin refers to a non-naturally occurring form of a defensin. These defensins may differ from defensins isolated from their natural sources in some engineered ways, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, etc. Variant sequences may be found in the sequences presented as SEQ ID NOs: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, e.g. on the basis of a subsequence thereof, and/or by introducing nucleotide substitutions which do not result in another amino acid sequence of the defensin encoded by the nucleotide sequence but which correspond to the codon usage of the host organism intended to produce the enzyme, or which result in a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al, 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that these substitutions can be made outside the critical regions for molecular function and still result in active defensins. Amino acid residues essential for the activity of defensins, and thus preferably amino acid residues that are not subject to substitution, can be identified according to methods known in the art, e.g., site-directed mutagenesis or alanine-partitioning mutagenesis (see, e.g., Cunningham and Wells (1989), Science 244: 1081-. In the latter technique, mutations are introduced at every positively charged residue in the molecule and the resulting mutant molecules are tested for antimicrobial activity to identify amino acid residues that are critical to the activity of the molecule. The site of interaction can also be determined by analysis of the three-dimensional structure, as determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al, (1992), Science 255: 306-.

The present invention also relates to polynucleotides encoding a defensin of the invention which hybridize under low stringency conditions, preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions, to the following sequences: (i) comprises the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, (ii) the mature peptide-encoding nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, or (iii) the complement of (i) or (ii); or allelic variants and subsequences thereof as defined herein (Sambrook et al, (1989), supra).

The present invention also relates to polynucleotides obtainable by: (a) hybridizing a population of DNA under low, medium-high, high or very high stringency conditions with: (i) comprises the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, (ii) the mature defensin-encoding nucleotide sequence of SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, or (iii) the complement of (i) or (ii); and (b) isolating the hybridized polynucleotide encoding the polypeptide having antimicrobial activity.

Nucleic acid construct:

the invention also relates to nucleic acid constructs comprising a polynucleotide encoding a defensin of the invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

Polynucleotides encoding the defensins of the invention can be manipulated in various ways to provide for expression of the defensin. Manipulation of the polynucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a defensin of the invention. The promoter sequence contains transcriptional control sequences that mediate the expression of defensins. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid construct of the invention in a filamentous fungal host cell are promoters from the genes for the following enzymes: aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic protease, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenaturosidase (WO00/56900), Fusarium venenatum (Daria) (WO00/56900), Fusarium venenatum (Quinn) (WO00/56900), Fusarium oxysporum trypsin-like protease (WO96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Aspergillus niger amyloglucosidase IV, Aspergillus niger alpha-amylase, Aspergillus niger neutral alpha-amylase, Aspergillus niger, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, and NA2-tpi promoter (a hybrid of promoters from Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase genes); and their mutant, truncated, and hybrid promoters.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the defensin. Any terminator which is functional in the host cell of choice may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the genes for the following enzymes: aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the defensin. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.

For filamentous fungal host cells, preferred polyadenylation sequences are derived from the genes for the following enzymes: aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a defensin and directs the encoded defensin into the cell's secretory pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted defensin. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region may be desirable where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the defensin. However, any signal peptide coding region that directs the expressed defensin into the secretory pathway of a host cell of choice may be used in the present invention.

For filamentous fungal host cells, an effective signal peptide coding region is one derived from the genes for the following enzymes: aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Cladosporium lanuginosum lipase.

In a preferred aspect, the signal peptide coding region is SEQ ID NO: 1, encoding nucleotides 1 to 69 of SEQ ID NO: 2 amino acids-55 to-33; SEQ ID NO: 3, which encodes seq id NO: 4 amino acids-48 to-29; SEQ ID NO: 5, which encodes SEQ id no: 6 amino acid-50 to-31; SEQ ID NO: 7, which encodes nucleotides 1 to 66 of SEQ ID NO: 8 amino acids-22 to-1; SEQ ID NO: 9, which encodes nucleotides 1 to 72 of SEQ ID NO: 10 amino acid-24 to-1; SEQ ID NO: 11, which encodes nucleotides 1 to 66 of SEQ ID NO: 12 amino acids-22 to-1; SEQ ID NO: 13, which encodes nucleotides 1 to 66 of SEQ ID NO: 14 amino acids-22 to-1; or SEQ ID NO: 15, which encodes nucleotides 1 to 54 of SEQ ID NO: 16 amino acids-26 to-9.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a pre-polypeptide (or in some cases as a zymogen (zymogen)). The propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for the following enzymes: bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and myceliophthora thermophila laccase (WO 95/33836).

In a preferred aspect, the propeptide coding region is SEQ ID NO: 1, which encodes nucleotides 70 to 165 of SEQ ID NO: 2 amino acid-32 to-1; SEQ ID NO: 3, which encodes nucleotides 61 to 144 of SEQ ID NO: 4 amino acid-28 to-1; SEQ ID NO: 5, which encodes nucleotides 61 to 150 of SEQ ID NO: 6 amino acid-30 to-1; or SEQ ID NO: 15, which encodes nucleotides 55 to 78 of SEQ ID NO: 16 amino acids-8 to-1.

When both the signal peptide and the propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences which allow the regulation of defensin expression relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, aspergillus niger glucoamylase promoter, and aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow amplification of a gene. In eukaryotic systems, these include the dihydrofolate reductase (dihydrofolate reductase) gene, which is amplified in the presence of methotrexate, and the metallothionein (metallothionein) gene, which is amplified with heavy metals. In these cases, the nucleotide sequence encoding the defensin will be operably linked to the regulatory sequence.

Expression vector:

the invention also relates to recombinant expression vectors comprising a polynucleotide encoding a defensin of the invention, a promoter and termination signals for transcription and translation. The various nucleic acid and control sequences described above may be combined to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding a defensin at these sites. Alternatively, the nucleotide sequence encoding the defensin of the invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector. In creating an expression vector, the coding sequence is placed in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means (means) to ensure self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together comprise the total DNA to be introduced into the genome of the host cell, or a transposon may also be used.

The vectors used to express the defensins of the invention preferably comprise one or more selectable markers that provide for easy selection of transformed cells. Selectable markers are genes whose products provide biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs (prototrophy to autotroph), and the like.

Examples of selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in aspergillus cells are the amdS and pyrG genes of aspergillus nidulans or aspergillus oryzae, and the bar gene of streptomyces hygroscopicus.

The vector preferably contains elements that allow the vector to be integrated into the genome of the host cell or allow the vector to replicate independently of the genome in the cell.

For integration into the host cell genome, the vector may rely on a polynucleotide sequence encoding a defensin or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may comprise additional nucleotide sequences for directing integration by homologous recombination into the host cell genome at the exact location in the chromosome. To increase the likelihood of integration at a precise location, the integrated elements should preferably comprise a sufficient number of nucleic acids, for example 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrated element can be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integrated elements may also be non-coding or coding nucleotide sequences. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replication gene (plasmid replicator) that mediates autonomous replication, which can function in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication useful in filamentous fungal cells are AMA1 and ANS (Gems et al (1991), Gene 98: 61-67; Cullen et al (1987) Nucl. acids Res.15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of a plasmid or vector comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide encoding a defensin of the invention may be inserted into the host cell to increase production of the gene product. The increase in copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including an amplifiable selectable marker gene with the polynucleotide sequence, such that the cell contains amplified copies of the selectable marker gene, and thereby is able to screen for additional copies of the polynucleotide by culturing the cell in the presence of the appropriate selectable agent.

Methods for ligating the above elements to construct the recombinant expression vectors of the invention are well known to those skilled in the art (see, e.g., Sambrook et al (1989), supra).

Filamentous fungal host cells:

the host cell (or organism) of The invention is a filamentous fungus, including filamentous forms of The phylum Ascomycota (Ascomycota), Basidiomycota (Basidiomycota), chylomycota (chytrimycota) and Zygomycota (Zygomycota) (as defined by Kirk p.m.et al, In, Ainsworth and Bisby's dictionary of The fungus, 9th edition, 2001, CAB International, Wallingford, UK). Filamentous fungi are characterized by a vegetative mycelium composed of chitin and glucan and/or other complex polysaccharides, vegetative growth being by extension of the mycelium. Preferred carbon catabolism is obligately aerobic.

In one embodiment, the filamentous fungal host cell (or organism) belongs to the order Eurotiales of the subdivision Ascomycota; preferably, it belongs more particularly to the family Trichocomaceae (trichococcaceae family).

In a more preferred embodiment, the filamentous fungal host cell (or organism) is, but is not limited to, a cell of the following species, or a sexual (teleomorph), an anamorphic (anamorph) or homonym (synonym) thereof: acremonium, Aspergillus, Emericella, Eurotium, Fusarium, Humicola, Mucor, myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma. In a still more preferred embodiment, the filamentous fungal host cell is an Aspergillus. In another still more preferred embodiment, the filamentous fungal host cell is Acremonium. In another still more preferred embodiment, the filamentous fungal host cell is Fusarium. In another still more preferred embodiment, the filamentous fungal host cell is a Humicola. In another still more preferred embodiment, the filamentous fungal host cell is a Mucor. In another still more preferred embodiment, the filamentous fungal host cell is a myceliophthora. In another still more preferred embodiment, the filamentous fungal host cell is a Neurospora. In another still more preferred embodiment, the filamentous fungal host cell is a Penicillium. In another still more preferred embodiment, the filamentous fungal host cell is a Thielavia. In another still more preferred embodiment, the filamentous fungal host cell is a strain of the genus Tolypocladium. In another yet more preferred embodiment, the filamentous fungal host cell is Trichoderma. In a most preferred embodiment, the filamentous fungal host cell is Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus aculeatus, Aspergillus niger or Aspergillus oryzae. In another preferred embodiment, the filamentous fungal host cell is a Fusarium (also known as Fusarium (section Fusarium)) of the group of discolorations (section disorders). For example, the filamentous fungal host cell may be Fusarium bactridioides, Fusarium graminearum, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, or Fusarium trichothecioides. In another preferred embodiment, the filamentous fungal host cell is a Fusarium strain of the group Elegans acuminata (section Elegans), e.g., Fusarium oxysporum. In another most preferred embodiment, the filamentous fungal host cell is Humicola insolens or Geotrichum gossypii. In another most preferred embodiment, the filamentous fungal host cell is Mucor miehei. In another most preferred embodiment, the filamentous fungal host cell is myceliophthora thermophila. In another most preferred embodiment, the filamentous fungal host cell is Neurospora crassa. In another most preferred embodiment, the filamentous fungal host cell is Penicillium purpurogenum or Penicillium funiculosum (WO 00/68401). In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris (Thielavia terrestris). In another most preferred embodiment, the Trichoderma cell is Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

In a specific embodiment, the filamentous fungal host cell is Aspergillus oryzae or Aspergillus niger.

In a preferred embodiment of the invention, the host cell is a protease deficient or protease minus strain.

For example, this may be the protease deficient strain Aspergillus oryzae JaL125 lacking the alkaline protease gene designated "alp". This strain is described in WO9735956(Novo Nordisk A/S) (application date 1997, 10/2) or European patent 429490(Genencor) (application date 1991, 6/5), or a TPAP-free host cell, in particular a strain of Aspergillus niger, which is disclosed in WO9614404(Novo Nordisk A/S) (application date 1996, 5/17). Furthermore, host cells with reduced production of transcriptional activator (prtT), in particular Aspergillus niger or Aspergillus oryzae, as described in WO0168864(Novo Nordisk A/S) (application date 2001, 9, 2), are specifically included in the present invention.

An intron:

eukaryotic genes can be interrupted by intervening sequences (introns) which must be modified in the precursor transcript to produce functional mRNA. This process of intron removal is known as pre-mRNA splicing. In general, the branch point sequence of an intron is necessary for splicing via the lasso (lariat) -forming intron. The splicing signal is present directly at the boundaries of the intron splice sites. The intron splice sites are typically bounded at their 5 'and 3' ends by consensus intron sequences GT and AG, respectively. Although there has been no report of 3 'splice sites other than AG, there are some exceptions to the 5' GT splice sites. For example, there are precedents for replacing GT at the 5' boundary with CT or GC. There is also a strong preference for the nucleotide base of the GT following, ANGT (where N is A, C, G or T) (mainly a or T in yeast species), but there is no apparent preference for any particular nucleotide preceding the GT splice site. The 3' splice site AG is preceded by a pyrimidine nucleotide base (Py), i.e., C or T.

The number of introns that can interrupt a fungal gene varies from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more introns. They may be distributed throughout the gene, or located at the 5 'or 3' end of the gene. In s.cerevisiae, the intron is located primarily at the 5' end of the gene. Introns are generally less than 1kb in size, and are generally less than 400bp in yeast, and less than 100bp in filamentous fungi.

The intron branch point sequence 5 '-TACTAAC-3' of Saccharomyces cerevisiae rarely occurs in introns of filamentous fungi. A sequence extension closely or loosely resembling tactac is seen at the equivalent point of the filamentous fungal intron, which has the general consensus NRCTRAC, where N is A, C, G or T, and R is a or G. For example, in the putative consensus sequence of both Neurospora crassa and Aspergillus nidulans, the fourth position T is invariant. In addition, nucleotides G, A and C predominate over 80% of positions 3, 6 and 7, respectively, although position 7 in A.nidulans is more flexible, with only 65% C. However, positions 1, 2, 5 and 8 are less stringent in Neurospora crassa and Aspergillus nidulans. Other filamentous fungi have similar branch point extensions at equivalent positions of their introns, but too few samples to discern any clear trends.

The method and the application are as follows:

in a first aspect, the present invention provides a recombinant filamentous fungal host cell comprising a nucleic acid construct comprising an exogenous nucleic acid sequence encoding a defensin and one or more intron sequences. The term "exogenous nucleic acid sequence" means a nucleic acid sequence introduced from the outside (from an exogenous source).

The filamentous fungal host cell may be capable of expressing (producing) a defensin in an amount of at least 150%, preferably 200%, more preferably 250%, and most preferably 300% of the amount obtained when using a nucleic acid construct, such as a cDNA sequence, without an intron sequence.

Filamentous fungal host cells may be grown in YPM growth medium for 3-5 days at 30-35 deg.C (degree Celsius) with appropriate agitation. This method and other suitable methods of culturing filamentous fungi are well known in the art. Filamentous fungal host cells may be used for recombinant production of defensins.

In a second aspect, the present invention provides a method of recombinantly producing a defensin in a filamentous fungal host cell comprising culturing a filamentous fungal host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences; and recovering the defensin peptide. Suitable recovery methods are well known in the art.

The invention also relates to the use of a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences for improving the recombinant expression level of a defensin in a filamentous fungal host cell.

The expression level of the defensin may be at least 50% higher, preferably at least 75% higher, more preferably at least 100% higher, even more preferably at least 125% higher, most preferably 150% higher and especially 200% higher than when using a nucleic acid construct, such as a cDNA sequence, which does not comprise an intron sequence. Alternatively, the expression level of the defensin may be at least 150%, preferably 200%, more preferably 250%, and most preferably 300% of the expression level obtained when using a nucleic acid construct, such as a cDNA sequence, without an intron sequence. Expression levels are measured in grams of defensin protein per liter of fermentation broth.

In one embodiment, the nucleic acid construct of the invention comprises 1, 2, 3, 4 or 5 intron sequences, preferably 1, 2, 3 or 4 intron sequences, more preferably 1, 2 or 3 intron sequences, even more preferably 1 or 2 intron sequences, and most preferably 1 intron sequence.

In another embodiment, the nucleic acid construct of the invention comprises a nucleic acid sequence encoding a defensin peptide and at least 1, preferably at least 2, more preferably at least 3, and most preferably at least 4 intron sequences.

The intron sequence may be located in the coding portion of the signal, propeptide or mature peptide of the nucleic acid construct of the present invention. When the nucleic acid construct comprises more than 1 intron, the introns may be located in different parts of the construct.

The intron sequence may be located in virtually any part of the defensin gene transcribed into mRNA.

The preparation method comprises the following steps:

the defensins of the invention can be incorporated into a number of pharmaceutical formulations for therapeutic administration. More specifically, the defensins of the present invention can be formulated into pharmaceutical compositions by combining with an appropriate pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semi-solid, liquid or gaseous form, for example, tablets, capsules, powders, granules, ointments (ointment), creams (cream), foams, solutions, suppositories (suppositoriy), injections, inhalants, gels, microspheres, lotions (position) and aerosols (aerosol). As such, administration of defensins can be performed in a variety of ways, including oral, buccal (buccal), rectal, parenteral (parenteral), intraperitoneal (intraepithelial), intradermal, transdermal, intrathoracic (intrathoracic), and the like. According to the invention, the defensin is systemic after administration.

The defensins of the invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.). In pharmaceutical dosage forms, defensins may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are exemplary only, and are not intended to be limiting in any way.

For oral formulations, the defensins can be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, e.g., in combination with conventional additives, e.g., lactose, mannitol, corn starch or potato starch; in combination with a binder, for example, crystalline cellulose, cellulose derivatives, acacia (acacia), corn starch or gelatin; in combination with a disintegrant (disintegrant), for example, corn starch, potato starch, or sodium carboxymethyl cellulose; in combination with lubricants, for example, talc or magnesium stearate; and if desired, in combination with diluents, buffers, wetting agents, preservatives and flavouring agents.

Formulations for injection can be formulated by dissolving, suspending or emulsifying the defensin in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonizing agents, suspending agents, emulsifiers, stabilizers and preservatives.

The defensins of the invention may be used in aerosol formulations for administration by inhalation. Defensins can be formulated in pressurized acceptable propellants (pressurized acceptable propellants), such as dichlorodifluoromethane, propane, nitrogen, and the like.

In addition, defensins may also be formulated into suppositories by mixing with various bases (bases), such as emulsified bases or water soluble bases. The defensins can also be administered rectally by suppository. Suppositories may include carriers, for example, cocoa butter, carbowax and polyethylene glycols, which are meltable at body temperature but solidify at room temperature.

Unit dosage forms for oral or rectal administration may be provided, for example syrups, elixirs and suspensions, wherein each dosage unit, for example a teaspoonful amount, a tablespoonful amount, tablet or suppository, contains a predetermined amount of a composition containing one or more defensins of the invention. Similarly, unit dosage forms for injection or intravenous administration may comprise a defensin of the invention in a composition such as a solution in sterile water, physiological saline or other pharmaceutically acceptable carrier.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human or animal subjects, each unit containing a predetermined quantity of a defensin of the invention calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specification (specification) of the unit dosage form of the invention depends on the particular defensin used and the effect to be achieved, as well as the pharmacodynamics (pharmacodynamides) associated with the defensin in the host.

Pharmaceutically acceptable excipients (e.g., carriers, adjuvants, carriers or diluents) are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, osmo-regulating agents, stabilizers, wetting agents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 picograms (pg) to 100 mg/kg of subject weight per administration. The usual dosage may be 2 to 6 times daily in a tablet form, or once daily in a time-release capsule or tablet form containing a proportionately higher amount of the active ingredient. The delayed release effect may be obtained by a capsule material that dissolves at different pH values, by a capsule that releases slowly with osmotic pressure, or by any other known means of controlled release.

One skilled in the art will readily appreciate that dosage levels may vary as a function of the particular defensin, the severity of the symptoms and the subject's susceptibility to side effects. Certain defensins are more potent than others. The preferred dosage for a given defensin will be readily determined by one skilled in the art by various methods. A preferred method is to measure the physiological potency (physiologcalpotency) of a given defensin.

The use of liposomes as delivery vehicles is an interesting approach. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the cells for a sufficient period of time to fuse, using various methods to maintain contact, e.g., separation, binding agents, etc. In one aspect of the invention, the liposomes are designed to aerosolize for pulmonary administration. Liposomes can be prepared with purified proteins or peptides that mediate membrane fusion, such as Sendai virus or influenza virus, and the like. The lipid may be any useful combination of known liposome-forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipids will typically be neutral or acidic lipids, such as cholesterol, phosphatidylserine, phosphatidylglycerol, and the like.

For the preparation of liposomes, the method described by Kato et al (Expression of lipids by surface antigen in adult liver. Co-Expression of DNA and nuclear by a amplified lipid method. J. bone. chem., February vol.266, p.3361-3364.ISSN 0021-9258) can be used. Briefly, the lipid comprising the peptide and the chamber composition are combined in a suitable aqueous medium, conveniently a saline medium, wherein the total solids will be in the range of about 1-10 weight percent. After a short period of vigorous stirring (about 5-60 seconds), the tube is placed in a warm water bath (about 25-40 ℃) and this cycle is repeated about 5-10 times. The composition is then sonicated for a convenient period of time (typically about 1 to 10 seconds) and may be further agitated by vortexing. The volume is then expanded by adding an aqueous medium, typically by about 1-2 volume increments, then shaken and cooled. This method allows the incorporation of high molecular weight molecules into the chamber.

Formulation with other active agents:

for use in the methods of the invention, the defensins of the invention may be formulated with other pharmaceutically active agents (e.g., steroids) which are well known in the art, particularly other antimicrobial agents. Other agents of interest include various antibiotics known in the art. The classes of antibiotics include penicillins such as penicillin G, penicillin V, dimethoxybenzene penicillin (methicillin), oxacillin (oxacillin), carbenicillin (carbenicillin), carbenicillin (nafcillin), ampicillin, and the like; penicillin combination beta-lactamase inhibitors, cephalosporins, such as cefaclor (cefaclor), cefazolin (cefazolin), cefuroxime (cefuroxime), cephalooxycarboxamide (moxalactam), and the like; carbapenems (carbapenems); monocyclolactams (monobactams); aminoglycosides (aminoglycosides); a tetracycline; macrolides (macrolides); lincomycin (lincomycins); polymyxins (polymyxins); sulfonamides (sulfonamides); quinolones (quinolones); chloramphenicol (cloramphenical); metronidazole (metronidazole); spectinomycin (spectinomycin); trimethoprim (trimethoprim); vancomycin (vancomycin) and the like.

Anti-mycotic agents (anti-mycotic agents) are also useful, including polyenes such as amphotericin B, nystatin; 5-fluorocarbonic acid (5-flucosyn); and azoles such as miconazole (miconazol), ketoconazole (ketoconazole), itraconazole (itraconazole), and fluconazole (fluconazol). Antituberculous drugs include isoniazid (isoniazid), ethambutol (ethambutol), streptomycin (streptomycin) and rifampin (rifampin). Cytokines may also be incorporated into the defensin formulations of the invention, such as interferon gamma, tumor necrosis factor alpha, interleukin 12, and the like.

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Example (b):

the chemicals used as buffers and substrates are at least reagent grade commercial products.

Example 1: expression of a defensin coding sequence comprising an intron in Aspergillus oryzae

The 361bp BamH1-Xho1 digested PCR product amplified from the P.nigricans (P.nigrella) cDNA library (SEQ ID NO: 6 from WO03044049, (Novo Nordisk A/S) (application date 2003, 5/30) was cloned into an Aspergillus expression vector as previously described in WO03/044049 to give plasmid pMT 2549. The intron sequence in the Plectasin (Plectasin) coding sequence of pMT2549 was modified by using standard in vitro mutagenesis and SOE to introduce a single additional base, thereby creating a restriction site in the intron. The resulting plectasin-containing (59bp) coding sequence is shown as SEQ ID NO: 17. the corresponding expression plasmid was designated pMT 2647.

pMT2647 was transformed into Aspergillus oryzae BECh2 as previously described in WO 03/044049. First, 14 independent transformants were re-isolated twice and grown on YPM medium (1% yeast extract, 2% bactopeptone and 2% maltose) and finally 10. mu.L of the sample was analysed on SDS gel as described previously in WO 03/044049. For some transformants, the amount of mycelial production, as shown by staining intensity, significantly exceeded the estimated maximum level of 50 mg/liter obtained under these growth conditions for Aspergillus oryzae transformants previously having an intron-free cDNA expression plasmid encoding mycelial (see WO 03/044049).

Although several of the 14 pMT2549 transformants appeared to produce higher levels of plectasin than the best 30 intron-free transformants previously analyzed in WO03/044049, it should be remembered that each acetamide-screened transformant represented separate transformation and integration events, which resulted in large differences in yield between the individual transformants. These yield differences are known to occur in non-homologous recombination-based systems and are generally considered to be the result of random integration of loci and the number of expression plasmids integrated. It was therefore decided to compare expression also in single copy expression vectors with defined locus integration (see example 2).

Example 2: expression of defensins in Aspergillus oryzae from defined Single copy integrants

In order to directly compare the expression of plectasin from intron-free cDNA with that of the intron-containing plectasin coding sequence in Aspergillus oryzae, these were transferred from pMT2548 (see WO03/044049) and pMT2549 (supra) to BamH1-Xba1 fragments of about 1.1kb (8.3kb), respectively, onto BamH1-Xba1 fragments of pJaL 485-based vectors (see example 3 of WO03008575(Novo Nordisk A/S) (application date 1/30/2003). For the screening effect, pJaL485 contained only a portion of the A.oryzae niaD gene encoding the C-terminal portion of the nitrate reductase. A host strain is used, such as the derivative JaL294, JaL507 (see example 8 of WO 03/008575), which contains a deletion in this part of the functional nitrate reductase niaD gene and therefore the ability to grow when nitrate is used as a nitrogen source can only be restored by homologous recombination. It has been found that the majority of transformants screened in this way are indeed single copy integrants resulting from a single homologous crossover. Tandem integrants at the niaD position do occur at a lower frequency. The intron-free and intron-containing pJaL 485-derived expression plasmids were designated pMT2777 and pMT2836, respectively. 4 transformants of Aspergillus oryzae JaL507 were screened for each of these plasmids and single copy transformed plasmids containing homologous integration in the niaD locus were shown by Southern analysis. The pMT 2777-derived Aspergillus oryzae transformant was named MT2882-2885 and the pMT 2836-derived transformant was named MT 2886-2889. Each of the transformants MT2882-2889 was grown in YPM as described above, and 10. mu.L of culture supernatant samples after 3 days and 7 days of growth were analyzed by PAGE SDS gel as described above. The results clearly show little distribution (spread) among transformants for each plasmid (as expected, since they should be independent but the same strain). On the other hand, the expression level in MT2886-2889 (containing introns) was significantly higher than that in MT2882-2885 (without introns).

On day 7, the relative plectasin levels of MT2886-2889 samples have also been determined by enzyme-linked immunosorbent assay (ELISA) (see example 4).

Example 3: increased defensin expression of genes comprising introns at different positions and/or different introns

To facilitate transfer of the sequences encoding mycelial variants from, for example, E.coli and Saccharomyces cerevisiae vectors to Aspergillus expression vectors, it was decided to relocate the mycelial intron from the position originally placed in the sequence encoding mature mycelial to the position in the coding sequence for the prepropeptide (prepro-peptide). To perform this relocation, mutations were introduced into the intron-free myceliophthora coding sequence by in vitro mutagenesis, resulting in the Mlun1 site located in the signal peptide coding sequence. It is only possible to introduce the MluN1 site by changing the amino acid in the signal peptide (Leu17 to Ala). According to commonly used signal prediction programs, it is predicted that such amino acid changes will not impair signal peptide function. This intron-free, Mlun 1-containing plectasin-encoding sequence is shown as SEQ ID NO: 18; the corresponding Aspergillus expression plasmid was designated pMT 2898.

An intron derived from the second intron of A.niger glucoamylase (see example 1 of WO03104457(NovoNordisk A/S) (application date 2003, 12/18) constructed as 55bp SnaB1-Pvu 2) and so that it can be excised from plasmid pMT2374 was inserted into the MluN1 site of pMT2898 to give pMT2899, in which the intron was confirmed to be inserted in the proper orientation. The sequence encoding plectasin of this intron-containing pMT2899 is shown as SEQ ID NO: 19.

furthermore, an intron originally present in the sequence encoding mature plectasin was prepared synthetically and allowed to move as a SnaB1-Pvu2 fragment in the same manner. This is only possible by changing the last two bases of the intron from T to C. This intron was also transferred to the MluN1 site of pMT2898 to give pMT2900, where the correct intron orientation was confirmed by sequencing. The myceliophthorne coding sequence of pMT2900 containing the intron is shown as SEQ ID NO: 20.

plasmids pMT2898, 2899 and 2900 were transformed into Aspergillus oryzae BECh2 and grown on acetamide for selection. Approximately 20 transformants with each plasmid were reisolated twice and grown on YPM medium as described above. It is evident from the SDS PAGE gels of the supernatants that the average mycelial expression level of the transformants with the constructs pMT2899 and pMT2900 containing introns is significantly higher than the average mycelial expression level of the transformants with the construct pMT2898 without introns. Furthermore, since the expression levels varied greatly between transformants selected with independent acetamide, it was decided to transfer the expression cassette of MT2898-2900 to the niaD-based expression vector derived from pJaL485, as described in example 2 above. The resulting expression plasmids were pMT2901, pMT2902 and pMT2903, corresponding to pMT2898, pMT2899 and pMT2900, respectively. pMT2901-2903 is transformed into niaD deficient host JaL507 as described above in example 2. Many transformants were reisolated for each transformation. Transformants were grown on YPM and supernatants were run on SDSPAGE gels as described above. Also in this case, the expression levels of constructs pMT2902 and pMT2903 containing introns were significantly higher than that of the transformants with construct pMT 2901. There is still a need for Southern analysis to demonstrate which transformants are indeed single copy integrants, but empirically, most nitrate-selected transformants in this setting (set up) can show up as single copy integrants even though tandem integration does occur. Two transformants were selected as representative strains per plasmid: pMT2901 (strains MT2946 and MT2947), pMT2902 (strains MT2948 and MT2949) and pMT2903 (strains MT2952 and MT 2953).

ELISA quantification of plectasin was also performed on MT2946-2949 (see example 4).

Example 4: competitive enzyme-linked immunosorbent assay-assessment of myceliophthorin yield in Aspergillus oryzae fermentation

Indirect competitive enzyme-linked immunosorbent assay was applied using rabbit polyclonal antibodies raised against purified plectasin to assess yield in aspergillus oryzae fermentation broth. This type of assay is a standard method generally applied to quantify a given protein/peptide, assuming that antisera have been generated.

The following materials and buffers were used:

-plectasin: defensins described in PCT application WO 03/044049;

-F96 MaxiSorp plate (Nunc, Cat. No.: 439454);

-F96 microwell plate (Nunc, Cat No.: 269787);

skim milk powder (Merck, Cat. No: 1.15363.0500);

tween20(Merck, Cat No.: 8.22184.0500);

-plectasin-specific rabbit polyclonal antibodies (Novazymes);

-porcine anti-rabbit immunoglobulin/HRP (DAKO, P0448);

TMB Plus, Ready-to-Go (KemEnTek, Cat. No.: 4390);

sulfuric acid (Merck, Cat. No: 1.00731.1000);

phosphate Buffered Saline (PBS), ph7.2, 1L:

8.00 grams of sodium chloride,

0.20 g of potassium chloride,

1.04 g of dipotassium hydrogen phosphate,

0.32 g of monopotassium phosphate;

-blocking buffer: phosphate buffer solution, 2% skim milk;

-wash buffer: phosphate buffer, 0.05% Tween 20;

-dilution buffer: phosphate buffer solution, 0.5% skim milk, 0.05% Tween 20;

briefly, undiluted and serial two-fold dilutions of culture broth (culture broth) were preincubated with 1: 1000 polyclonal antibody in dilution buffer. After 2 hours incubation at room temperature, the samples were transferred to Plectasin pre-coated (Plectasin pre-coated) plates (coated with 0.1 μ g/ml of Plectasin and the remaining binding blocked using blocking buffer). After incubation for 1 hour at room temperature, bound antibodies were detected using secondary antibodies (porcine anti-rabbit-HRP) and then using chromogens (TMBPlus). Detection of myceliomycin binding antibody is measured by absorbance at 450nm, which yields a titration curve for antibody binding.

Throughout the assay, the plates were washed with wash buffer and material dilutions (substance dilution) were made as described by the manufacturer (DAKO & kemnetek).

Explanation: no antibody binding to the wells was detected, representing complete binding (inhibition) of the antibody produced to the unknown fermentation broth; whereas the maximum absorption measured is equal to the inhibition of the missing competitor (unknown fermentation broth). In this way, we were able to assess the amount required to inhibit 50% of the binding to the wells (diluted broth). The results are described in the table below. The results were calculated relative to the yields measured for the fermentation broths without introns (MT2882, MT2883, MT2884 and MT 2885). The results are shown in Table 1.

The results show that by using the gene construct containing an intron, the expression level was increased to about 330% of the expression level obtained by using the gene construct without an intron.

TABLE 1

Intron	Culture ID	Relative yield	Average yield
				-	MT2882, day 7	106	100
-	MT2883, day 7	108
				-	MT2884, day 7	96
-	MT2885, day 7	91
				+	MT2886, day 7	335	330
+	MT2887, day 7	368
				+	MT2888, day 7	310
+	MT2889, day 7	320
				-	MT2946	77	89
-	MT2947	100
				+	MT2948	268	331
+	MT2949	394

Example 5: increased expression of defensins from amygdalina (e.amstelodami)

A defensin-encoding cDNA from P.avermitilis has been previously identified and designated as sanocin (Eurocin) (see example DK2005/000725(Novozymes A/S); or SEQ ID NO: 3). Expressing the cDNA in Aspergillus oryzae to obtain active Eurotin defensin peptide. To increase the expression level, an expression construct comprising a genomic DNA sequence (comprising two introns) was prepared.

Genomic DNA of D.amethystoides was prepared using standard methods for preparing fungal genomic DNA. Approximately 50ng of genomic DNA was used as template in the PCR reaction, with the following primers:

primer A:

TCTTGGATCCACCATGCACTTCACCAAGGTCTCC(SEQ ID NO：21)

and (3) primer B:

TCTTCTCGAGTTAGAAAGAACAGGTGCAGGTAC(SEQ ID NO：22)

10pmol of each primer was used in a reaction volume of 50. mu.L. The annealing temperature was 55 ℃ and extension at 72 ℃ for 1 minute. A total of 35 cycles were performed using the Expand High Fidelity PCR System (Roche). The PCR product was digested with BamH1 and Xho1, which cut in the overhang introduced by the PCR primers. The digests were run on a 2% agarose gel and bands of approximately 400bp were isolated. The isolated bands were ligated into plasmid pMT2786 digested with BamH1-Xho1 (see example 2 of PCT/DK 2005/000725) and transformed into E.coli MT173, which was selected for leucine prototrophy. The insert of BamH1-Xho1 was sequenced and proved to encode a prepro-bunyadin sequence corresponding to a previously characterized cDNA (see PCT/DK2005/000725 or SEQ ID NO: 3) but also comprising two intron sequences of 45 and 53 bases respectively. The sequence and structure of the BamH1-Xho1 insert is shown in SEQ ID NO: 23. the aspergillus expression plasmid containing the insert was designated pMT 2945.

pMT2945 was transformed into aspergillus oryzae BECh2, as described in example 1, and 20 transformants were reisolated and fermented as in example 1. The supernatant was also analyzed on SDS gel as described above. Parallel experiments were also carried out using transformants with the expression plasmid pMT2935 based on the bunyasu cDNA as before (see example 2 of PCT/DK 2005/000725).

Transformants based on the cDNA construct pMT2935 and the construct pMT2945 containing the intron both produced a clear band on SDS gel with a size corresponding to that of ascomycin.

Transformants based on the genomic construct pMT2945 comprising the intron were evaluated for 300-400% of the expression levels obtained for the generation of transformants based on the cDNA construct pMT 2935.

Example 6: identification of defensins using HMM profiles from PFAM database

Sequence analysis using hidden markov model profiles (HMM profiles) can be performed on the internet, online or locally on a computer, using HMMERs known to freely available software packages. The current version is HMMER2.3.2 since 10 months 2003.

HMM profiles are available from the well-known PFAM database. The current version is PFAM16.0 since 11 months 2004. For all computer platforms, HMMER and PFAM are available, for example, from the medical college of the University of Washington, san Louis (USA) (Washington University in St.Louis (USA), School of Medicine) (http:// pfam.wustly.ediu and http:// hmmer.wustly.ediu).

If the amino acid sequence of the query or fragment thereof is one of the following five PFAM families, the amino acid sequence is a defensin of the invention:

defensin _ β or "β defensin", accession number: PF 00711;

defensin _ propep or "defensin propeptide", accession No.: PF 00879;

defensin _1 or "mammalian defensin", accession number: PF 00323;

defensin _2 or "arthropod defensin", accession No.: PF 01097;

γ -thionin or "γ -thionin family", accession No.: PF 00304.

When the PFAM database is used online, or when the hmmpfam program (obtained from the HMMER software package) is used locally, an amino acid sequence belongs to the PFAM family according to the invention if it yields an E-value greater than 0.1 and a score greater than or equal to 0.

When sequence analysis is performed locally using the "hmmpfam" program, the HMM profiles need to be obtained (downloaded) from the PFAM database. There are two profiles for each family: hmm is used for global retrieval and xxx fs hmm is used for local retrieval ("xxx" is the name of a family). This resulted in a total of ten profiles for the five families described above.

These ten profiles can be used individually or combined (appended) into a single profile (using a text editor-the profile is an ASCII file) which can be named, for example, "defensein. The amino acid sequence of the query can then be evaluated by using the following command lines:

hmmpfam-E0.1 defensin.hmm sequence_file

-wherein "sequence _ file" is a file with the query amino acid sequence in any format identified by the HMMER software package.

If the score is greater than or equal to zero (0.0) and the E-value is greater than 0.1, then the amino acid sequence queried is a defensin of the invention.

PFAM database in Bateman et al, PFAM protein family database, nucl. D138-D141(BATEMAN,. The Pfam Protein family database. nucleic acid res., January vol.32, p.D138-D141. ISSN0305-1048.).

Sequence listing

<110> Novozymes corporation (Novozymes A/S)

<120> recombinant expression of defensins in filamentous fungi

<130>10789.204-WO

<160>24

<170>PatentIn version3.3

<210>1

<211>288

<212>DNA

<213> Pseudoplectania nigrella (Pseudoplectania nigrella)

<220>

<221>CDS

<222>(1)..(285)

<220>

<221> Signal peptide

<222>(1)..(69)

<220>

<221> mature peptide

<222>(166)..(285)

<400>1

atg caa ttt acc acc atc ctc tcc atc ggt atc acc gtc ttc gga ctt 48

Met Gln Phe Thr Thr Ile Leu Ser Ile Gly Ile Thr Val Phe Gly Leu

-55 -50 -45 -40

ctc aac acc gga gcc ttt gca gca ccc cag cct gtt ccc gag gct tac 96

Leu Asn Thr Gly Ala Phe Ala Ala Pro Gln Pro Val Pro Glu Ala Tyr

-35 -30 -25

gct gtt tct gat ccc gag gct cat cct gac gat ttt gct ggt atg gat 144

Ala Val Ser Asp Pro Glu Ala His Pro Asp Asp Phe Ala Gly Met Asp

-20 -15 -10

gcg aac caa ctt cag aaa cgt gga ttt gga tgc aat ggt cct tgg gat 192

Ala Asn Gln Leu Gln Lys Arg Gly Phe Gly Cys Asn Gly Pro Trp Asp

-5 -1 1 5

gag gat gat atg cag tgc cac aat cac tgc aag tct att aag ggt tac 240

Glu Asp Asp Met Gln Cys His Asn His Cys Lys Ser Ile Lys Gly Tyr

10 15 20 25

aag gga ggt tat tgt gct aag ggg ggc ttt gtt tgc aag tgt tac tag 288

Lys Gly Gly Tyr Cys Ala Lys Gly Gly Phe Val Cys Lys Cys Tyr

30 35 40

<210>2

<211>95

<212>PRT

<213> Pseudoplectania nigrella (Pseudoplectania nigrella)

<400>2

Met Gln Phe Thr Thr Ile Leu Ser Ile Gly Ile Thr Val Phe Gly Leu

-55 -50 -45 -40

Leu Asn Thr Gly Ala Phe Ala Ala Pro Gln Pro Val Pro Glu Ala Tyr

-35 -30 -25

Ala Val Ser Asp Pro Ghu Ala His Pro Asp Asp Phe Ala Gly Met Asp

-20 -15 -10

Ala Asn Gln Leu Gln Lys Arg Gly Phe Gly Cys Asn Gly Pro Trp Asp

-5 -1 1 5

Glu Asp Asp Met Gln Cys His Asn His Cys Lys Ser Ile Lys Gly Tyr

10 15 20 25

Lys Gly Gly Tyr Cys Ala Lys Gly Gly Phe Val Cys Lys Cys Tyr

30 35 40

<210>3

<211>273

<212>DNA

<213> Eurotium amstelodami (Eurotium amstelodami)

<220>

<22l>CDS

<222>(1)..(270)

<220>

<221> Signal peptide

<222>(1)..(60)

<220>

<221> mature peptide

<222>(145)..(270)

<400>3

atg cac ttc acc aag gtc tcc acc att ctt ttt acc atc ttc gcc gcc 48

Met His Phe Thr Lys Val Ser Thr Ile Leu Phe Thr Ile Phe Ala Ala

-45 -40 -35

ggc atc atg gct gct ccc acc gaa gga gtc cgt gag gaa gcc gcc cct 96

Gly Ile Met Ala Ala Pro Thr Glu Gly Val Arg Glu Glu Ala Ala Pro

-30 -25 -20

ggc cag gag gtt tac ccc gac gaa cct cct gct tct ctg acc aag cgt 144

Gly Gln Glu Val Tyr Pro Asp Glu Pro Pro Ala Ser Leu Thr Lys Arg

-15 -10 -5 -1

ggc ttc gga tgt cct ggt gat gcc tac cag tgc agt gaa cac tgc agg 192

Gly Phe Gly Cys Pro Gly Asp Ala Tyr Gln Cys Ser Glu His Cys Arg

1 5 10 15

gcc ctg ggc ggt gga cgc act gga gga tac tgt gct gga cct tgg tat 240

Ala Leu Gly Gly Gly Arg Thr Gly Gly Tyr Cys Ala Gly Pro Trp Tyr

20 25 30

ttg ggt cac cct acc tgc acc tgt tct ttc taa 273

Leu Gly His Pro Thr Cys Thr Cys Ser Phe

35 40

<210>4

<211>90

<212>PRT

<213> Eurotium amstelodami (Eurotium amstelodami)

<400>4

Met His Phe Thr Lys Val Ser Thr Ile Leu Phe Thr Ile Phe Ala Ala

-45 -40 -35

Gly Ile Met Ala Ala Pro Thr Glu Gly Val Arg Glu Glu Ala Ala Pro

-30 -25 -20

Gly Gln Glu Val Tyr Pro Asp Glu Pro Pro Ala Ser Leu Thr Lys Arg

-15 -10 -5 -1

Gly Phe Gly Cys Pro Gly Asp Ala Tyr Gln Cys Ser Glu His Cys Arg

1 5 10 15

Ala Leu Gly Gly Gly Arg Thr Gly Gly Tyr Cys Ala Gly Pro Trp Tyr

20 25 30

Leu Gly His Pro Thr Cys Thr Cys Ser Phe

35 40

<210>5

<211>273

<212>DNA

<213> white spruce (Picea glauca)

<220>

<221>CDS

<222>(1)..(270)

<220>

<221> Signal peptide

<222>(1)..(60)

<220>

<221> mature peptide

<222>(151)..(270)

<400>5

atg aag ttc acc atc tcc atc atc gcc gct ctc gct ttc ttc gcc cag 48

Met Lys Phe Thr Ile Ser Ile Ile Ala Ala Leu Ala Phe Phe Ala Gln

-50 -45 -40 -35

gga atc gtg gca gct cct gcg cct atc ccc gag gcc gcc gcg gtg gct 96

Gly Ile Val Ala Ala Pro Ala Pro Ile Pro Glu Ala Ala Ala Val Ala

-30 -25 -20

gcc cca gag gcc gag cca aag gcg tta gat gag ctt ccg gag ttg caa 144

Ala Pro Glu Ala Glu Pro Lys Ala Leu Asp Glu Leu Pro Glu Leu Gln

-15 -10 -5

aag cgt ggc ttt ggg tgc aac ggt tgg cct ttc gag gac gat gag cag 192

Lys Arg Gly Phe Gly Cys Asn Gly Trp Pro Phe Glu Asp Asp Glu Gln

-1 1 5 10

tgc cat aat cac tgc aag acc att cct ggt tat aag ggt ggc tac tgt 240

Cys His Asn His Cys Lys Thr Ile Pro Gly Tyr Lys Gly Gly Tyr Cys

15 20 25 30

gcc aac gtt ggc acc act tgc aag tgc tac tag 273

Ala Asn Val Gly Thr Thr Cys Lys Cys Tyr

35 40

<210>6

<211>90

<212>PRT

<213> white spruce (Picea glauca)

<400>6

Met Lys Phe Thr Ile Ser Ile Ile Ala Ala Leu Ala Phe Phe Ala Gln

-50 -45 -40 -35

Gly Ile Val Ala Ala Pro Ala Pro Ile Pro Glu Ala Ala Ala Val Ala

-30 -25 -20

Ala Pro Glu Ala Glu Pro Lys Ala Leu Asp Glu Leu Pro Glu Leu Gln

-15 -10 -5

Lys Arg Gly Phe Gly Cys Asn Gly Trp Pro Phe Glu Asp Asp Glu Gln

-1 1 5 10

Cys His Asn His Cys Lys Thr Ile Pro Gly Tyr Lys Gly Gly Tyr Cys

15 20 25 30

Ala Asn Val Gly Thr Thr Cys Lys Cys Tyr

35 40

<210>7

<211>189

<212>DNA

<213> Virginia gigas shell (Crassostra virginica)

<220>

<221>CDS

<222>(1)..(186)

<220>

<221> Signal peptide

<222>(1)..(66)

<220>

<221> mature peptide

<222>(67)..(186)

<400>7

atg aaa gtg ttt gta ctt ctt aca ata gca gta atg ctt atg gta tct 48

Met Lys Val Phe Val Leu Leu Thr Ile Ala Val Met Leu Met Val Ser

-20 -15 -10

gcc gac gtt gct ttg gcc ggc ttt ggc tgt cct ttg aac cgg tac cag 96

Ala Asp Val Ala Leu A1a Gly Phe Gly Cys Pro Leu Asn Arg Tyr Gln

-5 -1 1 5 10

tgt cac tca cat tgc caa tcc att ggt cgt aaa ggt gga tac tgt ggt 144

Cys His Ser His Cys Gln Ser Ile Gly Arg Lys Gly Gly Tyr Cys Gly

15 20 25

ggg tgg tgg agt ttt aca tgc aca tgc tac cgc act aaa aag tag 189

Gly Trp Trp Ser Phe Thr Cys Thr Cys Tyr Arg Thr Lys Lys

30 35 40

<210>8

<211>62

<212>PRT

<213> Virginia gigas shell (Crassostra virginica)

<400>8

Met Lys Val Phe Val Leu Leu Thr Ile Ala Val Met Leu Met Val Ser

-20 -15 -10

Ala Asp Val Ala Leu Ala Gly Phe Gly Cys Pro Leu Asn Arg Tyr Gln

-5 -1 1 5 10

Cys His Ser His Cys Gln Ser Ile Gly Arg Lys Gly Gly Tyr Cys Gly

15 20 25

Gly Trp Trp Ser Phe Thr Cys Thr Cys Tyr Arg Thr Lys Lys

30 35 40

<210>9

<211>189

<212>DNA

<213> Gibbs Buthus martensii Karsch (Mesobuthus gibbosus)

<220>

<221>CDS

<222>(1)..(186)

<220>

<221> Signal peptide

<222>(1)..(72)

<220>

<221> mature peptide

<222>(73)..(186)

<400>9

atg aaa acc att gta ctt ctt ttc gtg ttg gct tta gta ttc tgc act 48

Met Lys Thr Ile Val Leu Leu Phe Val Leu Ala Leu Val Phe Cys Thr

-20 -15 -10

ctt gaa atg gga atg gtg gaa gcc gga ttc ggt tgt cca ttc aat caa 96

Leu Glu Met Gly Met Val Glu Ala Gly Phe Gly Cys Pro Phe Asn Gln

-5 -1 1 5

gga aga tgt cac aga cat tgt cga agt att cgt cga aga gga gga tat 144

Gly Arg Cys His Arg His Cys Arg Ser Ile Arg Arg Arg Gly Gly Tyr

10 15 20

tgc gat gga ttt ttg aaa caa aga tgt gtt tgt tat cgg aga taa 189

Cys Asp Gly Phe Leu Lys Gln Arg Cys Val Cys Tyr Arg Arg

25 30 35

<210>10

<211>62

<212>PRT

<213> Gibbs Buthus martensii Karsch (Mesobuthus gibbosus)

<400>10

Met Lys Thr Ile Val Leu Leu Phe Val Leu Ala Leu Val Phe Cys Thr

-20 -15 -10

Leu Glu Met Gly Met Val Glu Ala Gly Phe Gly Cys Pro Phe Asn Gln

-5 -1 1 5

Gly Arg Cys His Arg His Cys Arg Ser Ile Arg Arg Arg Gly Gly Tyr

10 15 20

Cys Asp Gly Phe Leu Lys Gln Arg Cys Val Cys Tyr Arg Arg

25 30 35

<210>11

<211>198

<212>DNA

<213> Pacific oyster (Crassostra gigas)

<220>

<221>CDS

<222>(1)..(195)

<220>

<221> Signal peptide

<222>(1)..(66)

<220>

<221> mature peptide

<222>(67)..(195)

<400>11

atg aaa gta ttc gtt ctt tta aca cta gct gtc ctt ctg atg gtt tct 48

Met Lys Val Phe Val Leu Leu Thr Leu Ala Val Leu Leu Met Val Ser

-20 -15 -10

gca gac atg gct ttt gct gga ttt ggg tgt ccg ggt aac cag tta aag 96

Ala Asp Met Ala Phe Ala Gly Phe Gly Cys Pro Gly Asn Gln Leu Lys

-5 -1 1 5 10

tgc aac aat cac tgc aag tcc att agt tgt aga gcg ggc tac tgt gat 144

Cys Asn Asn His Cys Lys Ser Ile Ser Cys Arg Ala Gly Tyr Cys Asp

15 20 25

gca gcc acg ctc tgg tta aga tgt aca tgt acc gat tgt aat gga aag 192

Ala Ala Thr Leu Trp Leu Arg Cys Thr Cys Thr Asp Cys Asn Gly Lys

30 35 40

aag taa 198

Lys

<210>12

<211>65

<212>PRT

<213> Pacific oyster (Crassostra gigas)

<400>12

Met Lys Val Phe Val Leu Leu Thr Leu Ala Val Leu Leu Met Val Ser

-20 -15 -10

Ala Asp Met Ala Phe Ala Gly Phe Gly Cys Pro Gly Asn Gln Leu Lys

-5 -1 1 5 10

Cys Asn Asn His Cys Lys Ser Ile Ser Cys Arg Ala Gly Tyr Cys Asp

15 20 25

Ala Ala Thr Leu Trp Leu Arg Cys Thr Cys Thr Asp Cys Asn Gly Lys

30 35 40

Lys

<210>13

<211>195

<212>DNA

<213> Virginia gigas shell (Crassostra virginica)

<220>

<221>CDS

<222>(1)..(192)

<220>

<221> Signal peptide

<222>(1)..(66)

<220>

<221> mature peptide

<222>(67)..(192)

<400>13

atg aaa gtg ttt gtt ctt cta aca ata gct gtc atg ctt ttg gta tct 48

Met Lys Val Phe Val Leu Leu Thr Ile Ala Val Met Leu Leu Val Ser

-20 -15 -10

gcc gat gta gct act gca gat aac gga tgt ccc cgt cgt ccg aga atc 96

Ala Asp Val Ala Thr Ala Asp Asn Gly Cys Pro Arg Arg Pro Arg Ile

-5 -1 1 5 10

tgt cac aat cgg tgc ata tac aaa ggt cgt aga ggc gga aaa tgt gtc 144

Cys His Asn Arg Cys Ile Tyr Lys Gly Arg Arg Gly Gly Lys Cys Val

15 20 25

gga aag tgg aga agc tta tgc gaa tgc atc tac cca tcg aag gcc ggg 192

Gly Lys Trp Arg Ser Leu Cys Glu Cys Ile Tyr Pro Ser Lys Ala Gly

30 35 40

tga 195

<210>14

<211>64

<212>PRT

<213> Virginia gigas shell (Crassostra virginica)

<400>14

Met Lys Val Phe Val Leu Leu Thr Ile Ala Val Met Leu Leu Val Ser

-20 -15 -10

Ala Asp Val Ala Thr Ala Asp Asn Gly Cys Pro Arg Arg Pro Arg Ile

-5 -1 1 5 10

Cys His Asn Arg Cys Ile Tyr Lys Gly Arg Arg Gly Gly Lys Cys Val

15 20 25

Gly Lys Trp Arg Ser Leu Cys Glu Cys Ile Tyr Pro Ser Lys Ala Gly

30 35 40

<210>15

<211>207

<212>DNA

<213> Aspergillus oryzae (Aspergillus oryzae)

<220>

<221>CDS

<222>(1)..(207)

<220>

<221> Signal peptide

<222>(1)..(54)

<220>

<221> mature peptide

<222>(79)..(207)

<400>15

atg aaa ctt ctg acg gtc gcc ttt tcc ctt ctt ctt ctc ggg caa gtc 48

Met Lys Leu Leu Thr Val Ala Phe Ser Leu Leu Leu Leu Gly Gln Val

-25 -20 -15

cat gcc agt cct ttg gta ctc gac aaa agg tct tcc tgc cag ttg ggt 96

His Ala Ser Pro Leu Val Leu Asp Lys Arg Ser Ser Cys Gln Leu Gly

-10 -5 -1 1 5

gac gtc tgg gac ctc aat gct gca gac gcc gcc tgc agc gct tcg tgt 144

Asp Val Trp Asp Leu Asn Ala Ala Asp Ala Ala Cys Ser Ala Ser Cys

10 15 20

gcc att caa cac ggc gac aaa cac ggc gga cac tgc gat aag aac aag 192

Ala Ile Gln His Gly Asp Lys His Gly Gly His Cys Asp Lys Asn Lys

25 30 35

gtc tgc gtc tgc aat 207

Val Cys Val Cys Asn

40

<210>16

<211>69

<212>PRT

<213> Aspergillus oryzae (Aspergillus oryzae)

<400>16

Met Lys Leu Leu Thr Val Ala Phe Ser Leu Leu Leu Leu Gly Gln Val

-25 -20 -15

His Ala Ser Pro Leu Val Leu Asp Lys Arg Ser Ser Cys Gln Leu Gly

-10 -5 -1 1 5

Asp Val Trp Asp Leu Asn Ala Ala Asp Ala Ala Cys Ser Ala Ser Cys

10 15 20

Ala Ile Gln His Gly Asp Lys His Gly Gly His Cys Asp Lys Asn Lys

25 30 35

Val Cys Val Cys Asn

40

<210>17

<211>362

<212>DNA

<213> Artificial

<220>

<223> myceliophthora encoding sequence having intron (see example 1)

<400>17

ggatccacca tgcaatttac caccatcctc tccatcggta tcaccgtctt cggacttctc 60

aacaccggag cctttgcagc accccagccg gtacccgagg cttacgctgt ttctgatccc 120

gaggctcatc ctgacgattt tgctggtatg gatgcgaacc aacttcagaa acgtggattt 180

ggatgcaatg gtccttggga tgaggatgat atgcagtgcc acaagtaaga atcacttata 240

actagtagat taagccaaga gtattggaac tgatgataaa tagtcactgc aagtctatta 300

agggttacaa gggaggttat tgtgctaagg ggggctttgt ttgcaagtgt tactagctcg 360

ag 362

<210>18

<211>303

<212>DNA

<213> Artificial

<220>

<223> sequence encoding plectasin comprising MluN1 without introns (see example 3)

<400>18

ggatccacca tgcaatttac caccatcctc tccatcggta tcaccgtctt cggactggcc 60

aacaccggag cctttgcagc accccagccg gtacccgagg cttacgctgt ttctgatccc 120

gaggctcatc ctgacgattt tgctggtatg gatgcgaacc aacttcagaa acgtggattt 180

ggatgcaatg gtccttggga tgaggatgat atgcagtgcc acaatcactg caagtctatt 240

aagggttaca agggaggtta ttgtgctaag gggggctttg tttgcaagtg ttactagctc 300

gag 303

<210>19

<211>358

<212>DNA

<213> Artificial

<220>

<223> Mycoplanin-encoding sequence having pMT2899 intron (see example 3)

<400>19

ggatccacca tgcaatttac caccatcctc tccatcggta tcaccgtctt cggactgggt 60

atgtacacca cccccttgcg tctgatctgt gacatatgta gctgactggt cagccaacac 120

cggagccttt gcagcacccc agccggtacc cgaggcttac gctgtttctg atcccgaggc 180

tcatcctgac gattttgctg gtatggatgc gaaccaactt cagaaacgtg gatttggatg 240

caatggtcct tgggatgagg atgatatgca gtgccacaat cactgcaagt ctattaaggg 300

ttacaaggga ggttattgtg ctaagggggg ctttgtttgc aagtgttact agctcgag 358

<210>20

<211>362

<212>DNA

<213> Artificial

<220>

<223> sequence encoding plectasin of pMT2900 (see example 3)

<400>20

ggatccacca tgcaatttac caccatcctc tccatcggta tcaccgtctt cggactgggt 60

aagaatcact tataactagt agattaagcc aagagtattg gaactgatga taaacagcca 120

acaccggagc ctttgcagca ccccagccgg tacccgaggc ttacgctgtt tctgatcccg 180

aggctcatcc tgacgatttt gctggtatgg atgcgaacca acttcagaaa cgtggatttg 240

gatgcaatgg tccttgggat gaggatgata tgcagtgcca caatcactgc aagtctatta 300

agggttacaa gggaggttat tgtgctaagg ggggctttgt ttgcaagtgt tactagctcg 360

ag 362

<210>21

<211>34

<212>DNA

<213> Artificial

<220>

<223> primer A (see example 5)

<400>21

tcttggatcc accatgcact tcaccaaggt ctcc 34

<210>22

<211>33

<212>DNA

<213> Artificial

<220>

<223> primer B (see example 5)

<400>22

tcttctcgag ttagaaagaa caggtgcagg tac 33

<210>23

<211>386

<212>DNA

<213> Eurotium amstelodami (Eurotium amstelodami)

<220>

<221>CDS

<222>(10)..(195)

<220>

<221> Signal peptide

<222>(10)..(69)

<220>

<221> mature peptide

<222>(154)..(377)

<220>

<221> Intron

<222>(196)..(240)

<220>

<221>CDS

<222>(241)..(305)

<220>

<221> Intron

<222>(306)..(358)

<220>

<221>CDS

<222>(359)..(377)

<400>23

ggatccacc atg cac ttc acc aag gtc tcc acc att ctt ttt acc atc ttc 51

Met His Phe Thr Lys Val Ser Thr Ile Leu Phe Thr Ile Phe

-45 -40 -35

gcc gcc ggc atc atg gct gct ccc acc gaa gga gtc cgt gag gaa gcc 99

Ala Ala Gly Ile Met Ala Ala Pro Thr Glu Gly Val Arg Glu Glu Ala

-30 -25 -20

gcc cct ggc cag gag gtt tac ccc gac gaa cct cct gct tct ctg acc 147

Ala Pro Gly Gln Glu Val Tyr Pro Asp Glu Pro Pro Ala Ser Leu Thr

-15 -10 -5

aag cgt ggc ttc gga tgt cct ggt gat gcc tac cag tgc agt gaa cac 195

Lys Arg Gly Phe Gly Cys Pro Gly Asp Ala Tyr Gln Cys Ser Glu His

-1 1 5 10

gtatgtcctg ctgctatcta gagtgaatga agctaatgaa tatag tgc agg gcc ctg 252

Cys Arg Ala Leu

15

ggc ggt gga cgc act gga gga tac tgt gct gga cct tgg tat ttg ggt 300

Gly Gly Gly Arg Thr Gly Gly Tyr Cys Ala Gly Pro Trp Tyr Leu Gly

20 25 30

cac cc gtatgttgcc tagatattta attgtttata gtcctttact gatgagctta 355

His Pro

35

tag t acc tgc acc tgt tct ttc taactcgag 386

Thr Cys Thr Cys Ser Phe

40

<210>24

<211>90

<212>PRT

<213> Eurotium amstelodami (Eurotium amstelodami)

<400>24

Met His Phe Thr Lys Val Ser Thr Ile Leu Phe Thr Ile Phe Ala Ala

-45 -40 -35

Gly Ile Met Ala Ala Pro Thr Glu Gly Val Arg Glu Glu Ala Ala Pro

-30 -25 -20

Gly Gln Glu Val Tyr Pro Asp Glu Pro Pro Ala Ser Leu Thr Lys Arg

-15 -10 -5 -1

Gly Phe Gly Cys Pro Gly Asp Ala Tyr Gln Cys Ser Glu His Cys Arg

1 5 10 15

Ala Leu Gly Gly Gly Arg Thr Gly Gly Tyr Cys Ala Gly Pro Trp Tyr

20 25 30

Leu Gly His Pro Thr Cys Thr Cys Ser Phe

35 40

Claims (15)

1. A recombinant filamentous fungal host cell comprising a nucleic acid construct comprising an exogenous nucleic acid sequence encoding a defensin and one or more intron sequences.

2. The host cell of claim 1, which is an Aspergillus host cell.

3. An aspergillus host cell according to claim 2, which is an aspergillus niger or aspergillus oryzae host cell.

4. The host cell of any one of claims 1-3, wherein the defensin is an alpha-defensin, beta-defensin, theta-defensin, arthropod defensin, insect defensin, or plant defensin.

5. The host cell of any one of claims 1-4, which is capable of producing a defensin in an amount of at least 150% of the amount obtained when using a nucleic acid construct without an intron sequence.

6. A method of recombinantly producing a defensin in a filamentous fungal host cell comprising culturing a filamentous fungal host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences; and recovering the defensin peptide.

7. The method of claim 6, wherein the filamentous fungal host cell is an Aspergillus host cell.

8. The method of claim 7, wherein the Aspergillus host cell is an Aspergillus niger or Aspergillus oryzae host cell.

9. The method of any one of claims 6-8, wherein the defensin is an alpha-defensin, beta-defensin, theta-defensin, arthropod defensin, insect defensin, or plant defensin.

10. The method of any one of claims 6-9 which results in production of defensin in an amount of at least 150% of the amount obtained when using a nucleic acid construct without intron sequences.

11. Use of a nucleic acid construct comprising a nucleic acid sequence encoding a defensin peptide and one or more intron sequences to improve the recombinant expression level of a defensin in a filamentous fungal host cell.

12. The use of claim 11, wherein the filamentous fungal host cell is an Aspergillus host cell.

13. The use of claim 12, wherein the Aspergillus host cell is an Aspergillus niger or Aspergillus oryzae host cell.

14. The use of any one of claims 11 to 13 wherein the defensin is an alpha-defensin, beta-defensin, theta-defensin, arthropod defensin, insect defensin or plant defensin.

15. The use of any one of claims 11 to 14, wherein the expression level of the defensin is at least 50% higher than if a nucleic acid construct not comprising an intron sequence was used.