Heterologous biosynthesis of nodulisporic acid

Abstract

Nodulisporic acid (NA) comprises a group of indole diterpenoids known for their potent insecticidal activity; however, the biosynthesis of NA by its natural producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult to achieve. Identification of genes responsible for NA production could enable optimization of the biosynthetic pathway to provide a means of obtaining NA for commercial applications. Obtaining useful amounts of NA using published fermentation methods is challenging, making gene knockout studies an undesirable approach to confirm gene function. Alternatively, heterologous gene expression of H. pulicicidum genes in more robust host species such as Penicillium paxilli provides a way to rapidly identify the function of genes that play a role in NA biosynthesis. In this work, we identified the function of four secondary metabolic genes essential for the biosynthesis of polysporic acid F (NAF) and reconstructed these genes in the genome of P. paxilli to enable heterologous production of NAF in this fungus.

Description

Technical Field

[0001] The present invention generally relates to novel polypeptides that catalyze at least one biochemical reaction to produce nodulisporic acid (NA), polynucleotides encoding these polypeptides, methods for manufacturing these polypeptides and polynucleotides, and methods for producing at least one NA by using these polypeptides and polynucleotides through heterologous expression in a licensed host. Background Art

[0002] Filamentous fungi produce a wide variety of interesting and useful chemical compounds. One such class of compounds, members of the indole diterpenoid (IDT) family, is noteworthy for its broad chemical diversity and associated biological activities, including anti-MRSA. 1 Anti-cancer 2，3 Anti-H1N1 4 Insecticide 5 and tremor 6 Its activity has garnered particular attention. NA ( Figure 1 (This refers to a group of bioactive quasi-paspalum-like IDTs produced by *Hypoxylon pulicicidum*, previously classified as a species of the genus *Nodulisporium*.) 7 Polysporic acid A (NAA)10 is particularly important because it exhibits potent insecticidal activity against blood-sucking arthropods, while showing no observable adverse effects on mammals. 5，8 .

[0003] Biosynthesis of NA from the naturally occurring bacterium *H. pulicicidum* is particularly difficult. Reported methods for NA biosynthesis require *H. pulicicidum* to be grown in complete darkness for 21 days in a highly nutrient-rich medium. 9 Due to the difficulty of NAA10 biosynthesis in *H. pulicicidum*, obtaining useful amounts of NAA10 using published fermentation methods is challenging, and the production of commercial-scale NAA10 is essentially impossible. Therefore, attempts have been made to chemically synthesize NAA10, leading to the discovery of the synthetic polysporic acid F(NAF)5a. 10 and polysporic acid D 7a 11 The mechanism is known, but the total synthesis of NAA 10 has not yet been achieved. 12 Therefore, there is a need in the art for novel methods of NAA 10 synthesis and / or biosynthesis that provide useful amounts of NAA 10.

[0004] The object of the present invention is to provide a polynucleotide encoding at least one enzyme in the NAA 10 biosynthetic pathway of H. pulicicidum, and / or to provide a method for producing at least one NA as an indole diterpenoid compound and / or a precursor for producing NAA 10 in a heterologous host using such a vector, and / or to at least provide a useful option for the public.

[0005] In this specification, references to patent specifications, other external documents, or other sources of information are generally made for the purpose of providing background to the discussion of the features of the invention. Unless otherwise stated, references to such external documents should not be construed as an admission that such documents or sources are prior art or part of common general knowledge in the art within any jurisdiction. Summary of the Invention

[0006] On one hand, the present invention relates to an isolated polypeptide comprising an amino acid sequence selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof.

[0007] On the other hand, the present invention relates to an isolated polynucleotide encoding a polypeptide or a functional variant or fragment thereof comprising an amino acid sequence selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56).

[0008] On the other hand, the present invention relates to an isolated polynucleotide, which is selected from nodW cDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), and nodD2. The nucleic acid sequences of the cDNA (SEQ ID NO: 29), nodD2 genomic DNA (SEQ ID NO: 28), nodD1 cDNA (SEQ ID NO: 32), nodD1 genomic DNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 genomic DNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ genomic DNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS genomic DNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI genomic DNA (SEQ ID NO: 54) have at least 70% nucleic acid sequence identity.

[0009] On the other hand, the present invention relates to a transcription unit (TU) comprising at least one isolated polynucleotide according to the present invention.

[0010] On the other hand, the present invention relates to a carrier that encodes the isolated polypeptide according to the present invention.

[0011] On the other hand, the present invention relates to a vector comprising an isolated nucleic acid sequence or TU according to the present invention.

[0012] On the other hand, the present invention relates to an isolated host cell comprising isolated polypeptides, isolated polynucleotides, TUs and / or vectors according to the present invention.

[0013] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising heterologously expressing at least one polypeptide, isolated nucleic acid sequence, TU or vector according to the present invention in isolated host cells.

[0014] On the other hand, the present invention relates to at least one NA, which is manufactured by the method described in the present invention.

[0015] On the other hand, the present invention relates to an isolated polypeptide or a functional fragment or variant thereof derived from Hypoxylon spp. that catalyzes a biochemical reaction in the biosynthetic pathway from 3-geranylgeranylindole (GGI)2 to NAA10.

[0016] On the other hand, the present invention relates to an isolated polynucleotide encoding at least one polypeptide or a functional variant or fragment thereof derived from Hypoxylon spp., which catalyzes a biochemical reaction in the biosynthetic pathway that produces NAA10 from GGI2.

[0017] On the other hand, the present invention relates to a method for manufacturing at least one Hypoxylon spp. polypeptide or a functional variant or fragment thereof, the method comprising heterologously expressing an isolated nucleic acid sequence or vector according to the present invention in isolated host cells.

[0018] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising heterologously expressing at least one polypeptide in isolated host cells that catalyzes a biochemical reaction in the biosynthetic pathway that produces NAA10 from GGI2.

[0019] On the other hand, the present invention relates to an isolated host cell that expresses at least one heterologous polypeptide that catalyzes the conversion of a substrate in the biosynthetic pathway from GGI 2 to NAA 10.

[0020] On the other hand, the present invention relates to an isolated host cell that produces, via heterologous expression, at least one polypeptide involved in the biosynthetic pathway from GGI 2 to NAA 10.

[0021] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising contacting a substrate comprising carbohydrates with recombinant cells transformed with nucleic acids, the transformation resulting in an increased activity level of the recombinant cells compared with cells before transformation of polypeptides selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or functional variants or fragments thereof, such that the substrate is metabolized into at least one NA.

[0022] On the other hand, the present invention relates to an isolated strain of Hypoxylon pulicicidum containing at least one heterologous nucleic acid sequence encoding an enzyme in the biosynthetic pathway that produces NAA10.

[0023] On the other hand, the present invention relates to an isolated strain of Hypoxylon pulicicidum that expresses at least two different GGPPS enzymes.

[0024] On the other hand, the present invention relates to an isolated strain of Hypoxylon pulicicidum containing genetic modifications that cause an increase in the biosynthesis of NAA10.

[0025] On the other hand, the present invention relates to a method for manufacturing NAA10, the method comprising expressing at least one heterologous nucleic acid sequence in Hypoxylon pulicicidum, wherein the at least one heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway that produces NAA10.

[0026] Various embodiments of the different aspects of the invention discussed above are also set forth in the following detailed description of the invention, but the invention is not limited thereto.

[0027] Other aspects of the invention will become apparent from the following description, given only by way of example, and with reference to the accompanying drawings. Attached Figure Description

[0028] The invention will now be described with reference to the figures in the accompanying drawings.

[0029] Figure 1 : A known collection of polysporic acid (NA).

[0030] Figure 2 : Branching points in the biosynthetic pathway of indole diterpenes (IDTs), leading to the diversity of IDT structures. Arrows indicate enzymatic steps in IDT biosynthesis.

[0031] Figure 3 HPLC analysis (271 nm) of extracts from *Penicillium paxilli* knockout (KO) strains (gray) indicates different *H. pulicicidum* (Nod) enzymes and / or *Penicillium paxilli* (Pax) enzymes (black (-)). Black X covers enzymes not expressed in *Penicillium paxilli* KO strains (traces ia, ii.a, iii.a, iv.a, and va). Newly expressed enzymes in *Penicillium paxilli* KO strains are depicted below the corresponding KO strains and near their UV traces (ib, ii.b, iii.b, iv.b, and vb). Notably, compounds eluted at the same retention time as emindole SB 4a are present, but as confirmed by the corresponding 406.31 ± 0.01 m / z EIC. Figure 5 , 6 And 9), emindole SB4a exists only in three traces (ii.b, iii.b, and vb). The traces correspond to extracts of the fungus as follows: ia = PN2290, ib = pKV27:PN2690, ii.a = PN2257, ii.b = pKV63:PN2257, iii.a = PN2250, iii.b = pSK66:PN2250, iv.a = PN2250, iv.b = pKV74:PN2250, va = PN2257, vb = pKV64:PN2257.

[0032] Figure 4 The extracted ion chromatogram of pKV27:PN2290 (nodC:ΔpaxC) shows the MS peak of muscarinic acid 6b (5.3 min, 436.248 ± 0.01 m / z).

[0033] Figure 5 The extracted ion chromatogram of pKV63:PN2257 (nodM:ApaxM) showed an MS peak of emindole SB 4a (19.3 min, 406.31 ± 0.01 m / z) but no peak of paspalum 4b (17.6 min, 422.305 ± 0.01 m / z).

[0034] Figure 6 The extracted ion chromatogram of pSK66:PN2250 (paxG, nodC, nodM and nodB: ΔPAX cluster) shows the MS peak of emindole SB 4a (19.3 min, 406.31 ± 0.01 m / z) but without the MS peak of pax 4b (17.6 min, 422.305 ± 0.01 m / z).

[0035] Figure 7 The extracted ion chromatogram of pKV74:PN2250 (paxG, paxC, paxM, nodB: ΔPAX cluster) shows the MS peak of pax 4b (17.6 min, 422.305 ± 0.01 m / z) but without the MS peak of emindole SB 4a (19.3 min, 406.31 ± 0.01 m / z).

[0036] Figure 8 A diagram depicting the predicted NA gene clusters (A) and NAF 5a biosynthetic pathway (B) from *H. pulicicidum*. Arrows indicate individual genes, and arrow decorations indicate gene function. The diagram is not precisely to scale and does not include exon / intron structures. Note that the gene clusters lack GGPPS that correspond to the first secondary metabolic step in IDT synthesis.

[0037] Figure 9 The extracted ion chromatograms of pKV64:PN2257 (nodM and nodW:ΔpaxM) show MS peaks for emindole SB4a (19.3 min, 406.31 ± 0.01 m / z) and NAF 5a (6.2 min, 436.284 ± 0.01 m / z).

[0038] Figure 10 The extracted ion chromatogram of pSK68:PN2250 (paxG, nodC, nodM, nodB and nodW: ΔPAX cluster) shows MS peaks for emindole SB 4a (19.3 min, 406.31 ± 0.01 m / z) and NAF 5a (6.2 min, 436.284 ± 0.01 m / z).

[0039] Figure 11 Overview of MIDAS Level 1 Cloning.

[0040] Figure 12 MIDAS module address system.

[0041] Figure 13 Overview of the MIDAS box.

[0042] Figure 14 The principle of MIDAS multi-genome assembly (3 levels).

[0043] Figure 15 Overview of the MIDAS format. Detailed Description of the Invention

[0045] definition

[0046] When used in this specification and claims, the term "comprising" means "consisting of at least part of"; that is, when interpreting statements that include "comprising" in this specification and claims, the feature beginning with that term in each statement must be present, but other features may also be present. Related terms such as "comprising" should be interpreted in a similar manner.

[0047] When used herein, the term “consistent with substantially” means the specified materials and steps as well as those that do not substantially affect the essential and novel features of the claimed invention.

[0048] When used herein, the term “consisting of” means the specified materials or steps of the claimed invention, excluding any elements, steps, or components not specified in the claim.

[0049] The terms “recognition site” and “restriction site” are used interchangeably and have the same meaning herein. When used herein to refer to a restriction enzyme, these terms mean one or more nucleic acid sequences of a polynucleotide that define the binding site of a given restriction enzyme on said polynucleotide.

[0050] The term "indole diterpenoid (IDT) compound" or "indole diterpenoid" refers to any compound derived from a precursor containing indole, preferably indole-3-glycerophosphate 1b and geranylgeranyl pyrophosphate (GGPP) 1a.

[0051] In some embodiments, the IDT compound is selected from GGI 2, emindole SB 4a and NAF 5a.

[0052] The term "genetic construct" refers to a polynucleotide molecule, typically double-stranded DNA, that has been coupled to another polynucleotide molecule. In a non-limiting example, a genetic construct is created by inserting a first polynucleotide molecule into a second polynucleotide molecule, for example, by restriction / linking as known in the art. In some embodiments, a genetic construct comprises, but is not limited to, a single polynucleotide module, at least two polynucleotide modules, or a series of multiple polynucleotide modules assembled in a single, continuous polynucleotide molecule (also referred to herein as a "multigene construct").

[0053] The term "genetic construct" refers to a polynucleotide molecule, typically double-stranded DNA, that has been coupled to another polynucleotide molecule. In a non-limiting example, a genetic construct is created by inserting a first polynucleotide molecule into a second polynucleotide molecule, for example, by restriction / linking as known in the art. In some embodiments, a genetic construct comprises, but is not limited to, a single polynucleotide module, at least two polynucleotide modules, or a series of multiple polynucleotide modules assembled in a single, continuous polynucleotide molecule (also referred to herein as a "multigene construct").

[0054] The genetic construct may contain essential elements that allow transcription of polynucleotide molecules and, optionally, translation of the transcripts into polypeptides. The polynucleotide molecules contained in and / or included in the genetic construct may be derived from the host cell, or may be derived from different cells or organisms, and / or may be recombinant polynucleotides. Once inside the host cell, the genetic construct may become integrated into the host chromosomal DNA. The genetic construct may be ligated into a vector.

[0055] When used herein, the term “transcription unit” (TU) refers to a polynucleotide containing a sequence of nucleotides encoding a single RNA molecule, including, but not limited to, all nucleotide sequences necessary for transcription of the single RNA molecule, including, but not limited to, the promoter, RNA-coding sequence, and terminator.

[0056] When used herein, the term "transcriptional unit module" (TUM) refers to a polynucleotide sequence containing nucleotides that encode a single RNA molecule or a portion thereof, or a protein-coding sequence (CDS) or a portion thereof, or a sequence element or a portion thereof that encodes ...

[0057] When used in this document, the term "multigene construct" means a genetic construct consisting of a polynucleotide containing at least two TUs.

[0058] When used in this article, the term “tag” means the nucleic acid sequence in a polynucleotide that encodes a selection tag or scoring tag.

[0059] When used herein, the term "selection marker" refers to a TU that, when introduced into a cell, provides the cell with at least one trait that allows selection of the cell based on the presence or absence of that trait. In one embodiment, the cell is selected based on the condition that it survives by killing cells that do not contain the at least one selection marker.

[0060] When used herein, the term "scoring tag" refers to a TU that, when introduced into a cell, provides the cell with at least one trait, allowing the cell to be scored on the basis of the presence or absence of that trait. In one embodiment, cells containing the TU are scored by identifying the cell phenotypically from a plurality of cells.

[0061] When used herein, the term “genetic element” refers to any polynucleotide sequence that is not a TU or does not form part of a TU. These polynucleotide sequences may include, but are not limited to, origins of replication of plasmids and viruses, centromeres, telomeres, repetitive sequences, sequences for homologous recombination, site-specific recombination sequences, and sequences that control DNA transfer between organisms.

[0062] When used herein, the term "vector" refers to any type of polynucleotide molecule that can be used to manipulate genetic material such that it can be amplified, replicated, manipulated, partially replicated, modified, and / or expressed, but is not limited thereto. In some embodiments, a vector can be used to transport polynucleotides contained therein into cells or organisms.

[0063] When used herein, the term "source vector" refers to a vector into which the polynucleotide sequence of interest can be cloned. In some embodiments, the polynucleotide sequence is TU and TUM as described herein. In some embodiments, the source vector is selected from plasmids, bacterial artificial chromosomes (BACs), bacteriophage artificial chromosomes (PACs), yeast artificial chromosomes (YACs), bacteriophages, phage particles, and granules. In some embodiments, the source vector containing the polynucleotide sequence of interest is referred to as an introductory clone. In some embodiments, the introductory clone may be used as a shuttle or final vector to receive other polynucleotide sequences.

[0064] When used herein, the term "shuttle vector" refers to a vector into which polynucleotide sequences of interest can be cloned and manipulated. In some embodiments, the polynucleotide sequences are TU and TUM as described herein. In some embodiments, the shuttle vector is selected from plasmids, BACs, PACs, YACs, phages, phage particles, and granules. In some embodiments, the shuttle vector containing the polynucleotide sequence of interest can be used as a final vector to receive other polynucleotide sequences.

[0065] When used herein, the term "final vector" refers to a vector into which the polynucleotide sequence of interest can be cloned. In some embodiments, the polynucleotide sequence is TU and TUM as described herein. In some embodiments, the final vector is selected from plasmids, BACs, PACs, YACs, phages, phage particles, and granules. In some embodiments, the final vector containing the polynucleotide sequence of interest is an introductory clone. In some embodiments, the introductory clone can be used as a final vector to accept other polynucleotide sequences.

[0066] When used herein, the term "polynucleotide" means any single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and includes, as non-limiting examples, coding and non-coding sequences of genes, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors, and modified polynucleotides. The designations for nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences should be understood in the same way.

[0067] When used herein, the term "gene" refers to a hereditary biological unit that replicates itself and is located at a specific location (locus) on a particular chromosome. In one embodiment, the particular chromosome is a eukaryotic or bacterial chromosome. The term bacterial chromosome may be used interchangeably herein with the term bacterial genome.

[0068] When used herein, the term "gene cluster" refers to a group of genes located closely together on the same chromosome whose products play a coordinated role in specific aspects of primary or secondary metabolism in the cell. In one instance, a gene cluster comprises a set of CDS whose products are all involved in a series of biochemical reactions that constitute a biosynthetic pathway or array that produces a given metabolite, particularly a secondary metabolite.

[0069] When used in this article, the term "secondary metabolite" refers to compounds that do not participate in primary metabolism, and therefore they are distinct from more general macromolecules such as proteins and nucleic acids that constitute the basic mechanisms of life.

[0070] When used to refer to enzyme activity, the term "in which...the enzyme is active" and its grammatical variations mean that the enzyme will perform its intended function, such as restriction endonucleases cutting nucleic acids at suitable restriction sites, and DNA ligases covalently linking two polynucleotides together.

[0071] When used herein, the term "endogenous" means a component of a cell, tissue, or organism that originates from or is naturally produced in that cell, tissue, or organism. An "endogenous" component can be any component, including, but not limited to, polynucleotides, polypeptides including nonribosomal polypeptides, fatty acids, or polyketides.

[0072] When used herein, the term "exogenous" means any component of a cell, tissue, or organism that is not derived from or naturally produced in that cell, tissue, or organism. An exogenous component may be, for example, a polynucleotide sequence that has been introduced into a cell, tissue, or organism, or a polypeptide expressed from that polynucleotide sequence in that cell, tissue, or organism.

[0073] When used herein to refer to a polynucleotide sequence according to the present invention, "naturally occurring" means a primitive polynucleotide sequence found in nature. For the purposes of this disclosure, a synthetic polynucleotide sequence consistent with a wild-type polynucleotide sequence is considered a naturally occurring sequence. For naturally occurring polynucleotide sequences, it is important that the actual sequence of the nucleotide bases constituting the polynucleotide is found or known in nature.

[0074] For example, wild-type polynucleotide sequences are naturally occurring polynucleotide sequences, but are not limited to this. Naturally occurring polynucleotide sequences also refer to variant polynucleotide sequences found in nature that differ from the wild type. Examples include allelic variants resulting from hybridization or horizontal gene transfer and naturally occurring recombinant polynucleotide sequences, but are not limited to this.

[0075] When used herein to refer to a polynucleotide sequence according to the invention, "non-naturally occurring" means a polynucleotide sequence not found in nature. Examples of non-naturally occurring polynucleotide sequences include, but are not limited to, artificially generated mutant and variant polynucleotide sequences produced through, for example, point mutations, insertions, or deletions. Non-naturally occurring polynucleotide sequences also include chemically evolved sequences. For non-naturally occurring polynucleotide sequences according to the invention, it is important that the true sequence of the nucleotide bases constituting the polynucleotide has not been found or is known in nature.

[0076] When used herein to refer to polynucleotides, the term "wild-type" refers to the naturally occurring, non-mutant form of a polynucleotide. A mutant polynucleotide means, but is not limited to, a polynucleotide that maintains mutations known in the art such as point mutations, insertions, deletions, substitutions, amplifications, or translocations.

[0077] When used in this article to refer to polypeptides, the term "wild-type" refers to the naturally occurring, non-mutant form of the polypeptide. Wild-type polypeptides are polypeptides that can be expressed from wild-type polynucleotides.

[0078] The term "coding sequence" or "open reading frame" (ORF) refers to the positive strand or cDNA sequence of genomic DNA that is capable of producing transcripts and / or peptides under the control of suitable regulatory sequences. The CDS is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct or expression cassette, the "coding sequence" (CDS) can be expressed when operatively linked to a promoter and / or other regulatory elements.

[0079] "Operable connection" means that the sequence to be expressed is placed under the control of a regulatory element.

[0080] When used herein, a “regulatory element” means any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector, genetic construct, or expression cassette, and includes promoters, transcription control sequences, translation control sequences, origins of replication, tissue-specific regulatory elements, time-regulatory elements, enhancers, polyadenylation signals, repressors, and terminators. The regulatory element may be “homological” or “heterologous” to the polynucleotide insert to be expressed from the genetic construct, expression cassette, or vector described herein. When the genetic construct, expression cassette, or vector described herein is present in a cell, the regulatory element may be “endogenous,” “exogenous,” “naturally occurring,” and / or “non-naturally occurring” relative to the cell.

[0081] The term "non-coding region" refers to the untranslated sequences upstream of the translation start site and downstream of the translation termination site. These sequences are also known as the 5'UTR and 3'UTR, respectively. These regions contain elements required for transcription initiation and termination and for the regulation of translation efficiency.

[0082] Terminators are sequences that terminate transcription and are located at the 3' untranslated end of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and have been found to have spatial regulatory functions in some cases.

[0083] The term "promoter" refers to a non-transcriptional cis-regulatory element upstream of a coding region that regulates the transcription of a polynucleotide sequence. A promoter contains a cis-initiation element that designates the transcription start site and a conserved frame. In a non-restrictive example, a bacterial promoter may contain a "Pribnow box" (also known as the -10 region) and other motifs that are bound by transcription factors and promote transcription. A promoter can be homologous or heterologous to the polynucleotide sequence to be expressed. When a polynucleotide sequence is to be expressed in a cell, the promoter can be endogenous or exogenous. As is known in the art, promoters can be constitutive, inducible, or regulatory promoters.

[0084] When used herein to refer to polynucleotide regulatory elements, "homologous" means that the polynucleotide regulatory element is original and naturally occurring. Homologous polynucleotide regulatory elements can be operatively linked to the polynucleotide of interest, such that the polynucleotide of interest can be expressed from a TU, genetic element, or vector according to the invention.

[0085] When used herein to refer to polynucleotides or polypeptides in a host organism, "homologous" means that the polynucleotide or polypeptide is a native and naturally occurring polynucleotide or a polynucleotide within the host organism. Homologous polynucleotides can be operatively linked to homologous or heterologous regulatory elements, such that homologous polypeptides can be expressed from a TU, genetic element, or vector containing the homologous polynucleotides described herein.

[0086] When used herein to refer to polynucleotides or polypeptides in a host organism, "introduced homologous" means that the polynucleotide or polypeptide is an intrinsic and naturally occurring polynucleotide or a polynucleotide within the host organism that has been introduced into the organism experimentally. The introduced homologous polynucleotide can be operatively linked to homologous or heterologous regulatory elements, enabling the homologous polypeptide to be expressed from a TU, genetic element, or vector containing the homologous polynucleotide described herein.

[0087] When used herein to refer to polynucleotide regulatory elements, "heterologous" means that the polynucleotide regulatory element is not native and naturally occurring. Heterologous polynucleotide regulatory elements typically do not have a CDS operatively linked to them. Heterologous regulatory elements can be operatively linked to the polynucleotide of interest, such that the polynucleotide of interest can be expressed from the vector, genetic construct, or expression cassette according to the invention. These promoters may include normal promoters accompanied by other genes, ORFs, or coding regions, and / or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cells.

[0088] When used herein to refer to polynucleotides or polypeptides in a host organism, "heterologous" means that the polynucleotide or polypeptide is not native to or naturally occurring in that host organism. Heterologous polynucleotides can be operatively linked to heterologous or homologous regulatory elements, enabling the heterologous polypeptide to be expressed from a TU, genetic element, or vector containing the heterologous polynucleotides described herein.

[0089] The term "heterologous expression" means the expression of a heterologous polypeptide in a host cell.

[0090] "Biochemical reactions in the biosynthetic pathway from GGI 2 to NAA 10" means one of the specific reactions catalyzed by one of the specific enzymes involved in the conversion of the substrate molecule GGI 2 to NAA 10 via the following intermediates: monoepoxide GGI 3a, emindole SB 4a, NAF 5a, NAE 6a, NAD 7a, NAC 8, NAB 9, and excluding similar enzymes in the host cell that may have similar functions but do not act on the intermediates specifically named above.

[0091] A “functional variant or fragment thereof” of a polypeptide is a subsequence of the polypeptide that performs the biological activity of the polypeptide or performs the required function and / or provides the three-dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of polypeptides such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or a functional polypeptide derivative thereof capable of performing the activity of the polypeptide.

[0092] When used herein to refer to a polynucleotide or polypeptide sequence, "isolated" describes a sequence that has been removed from its native cellular environment. Isolated molecules can be obtained by any method or combination of methods known and used in the art, including biochemical, recombinant, and synthetic techniques. The polynucleotide or polypeptide sequence can be prepared by at least one purification step.

[0093] When used herein to refer to cells or host cells, "isolated" describes cells or host cells that have been obtained or removed from an organism or its natural environment and subsequently maintained in a laboratory setting known in the art. The term covers both single cells themselves and cells or host cells contained in cell cultures, and may include a single cell or a single host cell.

[0094] When used in this document to refer to fungal host cells, the term "isolated host cell" encompasses the single cells of unicellular fungi and the hyphae and mycelia of filamentous fungi, including septate and unseptate forms.

[0095] The term "recombinant" refers to a polynucleotide sequence that is taken from and / or recombined with sequences not present in its natural background. "Recombinant" polypeptide sequences are generated from "recombinant" polynucleotide sequences through translation.

[0096] When used herein, the term "variant" refers to a polynucleotide or polypeptide sequence that differs from a specifically designated sequence, wherein one or more nucleotide or amino acid residues are deleted, substituted, or added. A variant may be a naturally occurring allelic variant or a non-natural variant. Variants may originate from the same species or from other species and may encompass homologs, paralogs, and orthologs. In some embodiments, variants of the polypeptides used in the present invention may have the same or similar biological activities as the corresponding wild-type molecule, i.e., the parent polypeptide or polynucleotide.

[0097] In some embodiments, variants of the peptides described herein have biological activities similar to or substantially similar to their corresponding wild-type molecules. In some embodiments, the similarity refers to similar activity and / or binding specificity.

[0098] In some embodiments, variants of the peptides described herein have different biological activities than their corresponding wild-type molecules. In some embodiments, the difference is altered activity and / or binding specificity.

[0099] For polynucleotides and peptides, the term "variant" encompasses all forms of polynucleotides and peptides as defined herein.

[0100] The variant polynucleotide sequence preferably exhibits at least 50%, at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, and preferably at least 99% similarity to the sequence of the present invention. Consistency is found within a comparison window of at least 8 nucleotide positions, preferably at least 10 nucleotide positions, preferably at least 15 nucleotide positions, preferably at least 20 nucleotide positions, preferably at least 27 nucleotide positions, preferably at least 40 nucleotide positions, preferably at least 50 nucleotide positions, preferably at least 60 nucleotide positions, preferably at least 70 nucleotide positions, preferably at least 80 nucleotide positions, preferably across the entire length of the polynucleotide, using or identified by the method according to the invention.

[0101] Polynucleotide variants also encompass variants that exhibit similarity to one or more specifically designated sequences, which may retain the functional equivalence of those sequences and cannot be reasonably predicted to occur by random chance.

[0102] Polynucleotide sequence identity and similarity can be readily determined by those skilled in the art.

[0103] Variant polynucleotides also encompass polynucleotides that differ from the polynucleotide sequences described herein but encode polypeptides with similar activities to those encoded by the polynucleotides of this invention as a result of genetic code degeneracy. Sequence changes that do not alter the amino acid sequence of the polypeptide are “silent variants.” Codons for the same amino acids, other than ATG (methionine) and TGG (tryptophan), can be altered using techniques recognized in the art to, for example, optimize codon expression in a particular host organism.

[0104] The present invention also includes polynucleotide sequence changes that cause conserved substitutions of one or more amino acids in the encoded polypeptide sequence without significantly altering its biological activity. Those skilled in the art will recognize methods for producing phenotypic silencing amino acid substitutions (see, for example, Bowie et al., 1990, Science 247, 1306).

[0105] For polypeptides, the term "variant" also encompasses naturally occurring, recombinant, and synthetically produced polypeptides. The variant polypeptide sequence preferably exhibits at least 35%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, and preferably at least 99% similarity to the sequence of the present invention. Consistency is found within a comparison window of at least 2 amino acid positions, preferably at least 3 amino acid positions, preferably at least 4 amino acid positions, preferably at least 5 amino acid positions, preferably at least 7 amino acid positions, preferably at least 10 amino acid positions, preferably at least 15 amino acid positions, preferably at least 20 amino acid positions, preferably throughout the entire length of the polypeptide used or identified by the method according to the invention.

[0106] Peptide variants also encompass variants that exhibit similarity to one or more specifically designated sequences, which may retain the functional equivalence of those sequences and cannot be reasonably predicted to occur by random chance.

[0107] Peptide sequence identity and similarity can be readily determined by those skilled in the art.

[0108] Variant peptides include peptides in which one or more conserved amino acids or non-conserved substitutions, deletions, additions or insertions differ from the peptides described herein without affecting the bioactivity of the peptide.

[0109] Conservative substitutions typically involve replacing one amino acid with another amino acid that has similar characteristics, such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine, serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0110] Analysis of evolutionary biological sequences reveals that not all sequence variations are equally likely, reflecting at least in part the differences between conserved and non-conserved substitutions at the biological level. For example, some amino acid substitutions may occur frequently, while others are very rare. Evolutionary changes or substitutions of amino acid residues can be modeled using scoring matrices, also known as substitution matrices. These matrices are used in bioinformatics analyses to identify identity relationships between sequences and are known to those skilled in the art.

[0111] Other variants include peptides with modifications that affect peptide stability. These analogs may, for example, contain one or more non-peptide bonds (which replace peptide bonds) in the peptide sequence. Also included are analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids, or non-naturally occurring synthetic amino acids such as β or γ amino acids, and cyclic analogs.

[0112] Substitutions, deletions, additions, or insertions can be produced using mutagenesis methods known in the art. Those skilled in the art will recognize methods used to produce phenotypic silenced amino acid substitutions. See, for example, Bowie et al., 1990, Science 247, 1306.

[0113] When used herein, polypeptide may also refer to a polypeptide that has been modified during or after synthesis, such as by biotinylation, benzylation, glycosylation, phosphorylation, amidation, derivatization using blocking / protecting groups, etc. These modifications may improve the stability or activity of the polypeptide.

[0114] The term "regulation of polynucleotide or polypeptide expression" is intended to cover situations where the genomic DNA corresponding to the polynucleotide to be expressed according to the invention is modified, thereby resulting in the regulation of the expression of the polynucleotide or polypeptide of the invention. This modification of the genomic DNA can be achieved through genetic transformation or other methods known in the art for inducing mutations. The "regulated expression" may involve an increase or decrease in the amount of messenger RNA and / or polypeptide produced, and may also result in an increase or decrease in polypeptide activity due to changes in the sequence of the produced polynucleotide and polypeptide.

[0115] The term "regulating the activity of polynucleotides or peptides" is intended to cover situations where the genomic DNA corresponding to the polynucleotide to be expressed according to the present invention is modified, thereby resulting in the regulation of the expression of the polynucleotide or the regulation of the expression or activity of the peptide. The modification of the genomic DNA can be performed by genetic transformation or other methods known in the art for inducing mutations. The "regulated activity" can involve an increase or decrease in the amount of messenger RNA and / or peptide produced, and can also result in an increase or decrease in the functional activity of the peptide due to changes in the sequence of the produced polynucleotide and peptide.

[0116] References to the numerical ranges disclosed herein (e.g., 1 to 10) are also intended to include references to all related numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any reasonable range of numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are merely examples of the specific intent, and all possible combinations of numerical values ​​between the enumerated minimum and maximum values ​​should be considered as expressly stated in a similar manner in this application.

[0117] Detailed description

[0118] Since the identification of the biosynthetic pathway of IDT muscarinic acid 6b in Penicillium paxilli 6，13-16 Since then, the gene functions in seven other IDT biosynthetic pathways have been elucidated. 17-22 These IDT pathways share homologous genes encoding enzymes used in the first three steps of IDT biosynthesis. Figure 2 The pathway comprises (I) gerany-gerany pyrophosphate synthase (GGPPS), which converts farnesyl pyrophosphate and isopentyl pyrophosphate into GGPP 1a; (II) gerany-gerany transferase (GGT), which catalyzes the indole condensation of GGPP 1a and indole-3-glycerophosphate 1b to produce GGI 2; and (III) regioselective flavin adenine dinucleotide (FAD)-dependent cyclooxygenase, which produces mono- and / or bi-cyclooxidized GGI products 3a / 3b. In the fourth enzymatic step involving IDT cyclization, the pathway differentiates into four key branches, producing mono- and bi-oxygenated anti-Markovnikov-derived cyclic cores such as emindole SB 4a and paspalumine 4b, or mono- and bi-oxygenated Markovnikov-derived cyclic cores such as aflavinine 4c and emindole DB 4d. These cyclic cores are often further modified by decorative enzymes, resulting in the bioactivity diversity seen within the IDT.

[0119] NA is a bioactive IDT produced by Hypoxylon pulicicidum, among which NAA 10 is particularly important due to its highly effective insecticidal activity against blood-sucking arthropods and lack of mammalian toxicity. 5，8 However, as discussed in this paper, the direct synthesis of NAA 10 has not yet been achieved, and the biosynthetic production of this compound in quantities useful even at small-scale commercial levels is, if not entirely impossible, difficult to achieve.

[0120] Therefore, this invention generally relates to a series of genes isolated from the fungus *H. pulicicidum*, which combine to form a gene cluster mediating NA production, and to the use of this gene cluster to direct heterologous expression of NA in isolated host cells, preferably isolated fungal cells. Using a recently developed technique for manipulating gene sequences called the Modular Idempotent DNA Assembly System (MIDAS), the inventors have reconstructed the biosynthetic pathway for NAF 5a from *H. pulicicidum* in the optional fungal host *Penicillium paxilli*. The MIDAS platform and the methods using the MIDAS platform are described herein and throughout in the relevant patent application AU2017903955, which is incorporated herein by reference.

[0121] The inventors analyzed the genome sequence of *H. pulicicidum* and identified a gene cluster containing 15 predicted coding sequences (CDS): nodW cDNA (SEQ ID NO: 2) and genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5) and genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8) and genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11) and genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14) and genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17) and genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20) and genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23) and genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23) and genomic DNA (SEQ ID NO: 22), and nodY1 cDNA (SEQ ID NO: 19). NodD2 cDNA (SEQ ID NO: 26) and genomic DNA (SEQ ID NO: 25), nodD1 cDNA (SEQ ID NO: 32) and genomic DNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35) and genomic DNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38) and genomic DNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49) and genomic DNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55) and genomic DNA (SEQ ID NO: 54), which are expected to encode an enzyme essential for the biosynthesis of NAA10.

[0122] The boundaries of this gene cluster were determined by identifying genes flanking the equivalent genomic loci in another Hypoxylon strain that does not produce polysporic acid, exhibiting high similarity and co-linear organization. Details of the predicted genes in these clusters and their proposed functions are shown in Table 1. Seven genes in this cluster are homologous to genes found in other IDT biosynthetic gene clusters (Tables 2-6). The protein products of the seven predicted genes homologous to IDT biosynthetic genes from other fungi have at least 35% amino acid identity with their homologs in the PAX cluster of *Penicillium paxilli*, the JAN cluster of *Penicillium janthinellum*, and / or the PEN cluster of *Penicillium crustosum*, and include GGT (NodC (SEQ ID NO: 24)); two FAD-dependent oxidases (NodM (SEQ ID NO: 12) and NodO (SEQ ID NO: 18)), an IDT cyclase (NodB (SEQ ID NO: 15)), two isoprenyltransferases (NodD2 (SEQ ID NO: 30) and NodD1 (SEQ ID NO: 33)), and a cytochrome P450 oxygenase (NodR (SEQ ID NO: 6)). The other seven speculative ORFs are predicted to encode four cytochrome P450 oxygenases (NodW (SEQ ID NO: 3), NodX (SEQ ID NO: 9), NodJ (SEQ ID NO: 21), and NodZ (SEQ ID NO: 39)), a pair of paralogous FAD-dependent oxygenases (NodY1 (SEQ ID NO: 27) and NodY2 (SEQ ID NO: 36)), and two gene products with unknown functions that may be involved in NA biosynthesis (NodS (SEQ ID NO: 50) and NodI (SEQ ID NO: 56)). These are related to the TER gene cluster from *Chaunopycnisalba* (*Tolypocladium album*) responsible for terpendole biosynthesis. 17 Similarly, the NOD clusters do not appear to contain a secondary metabolite-specific GGPPS gene. Notably, the inventors identified only one gene encoding GGPPS in the genome of *H. pulicicidum*, and the amino acid sequence of its predicted protein product, its exon / intron structure, and its location outside the identified cluster strongly suggest that it is responsible for a primary metabolic function similar to ggs1 in *P. paxilli*. 23 .

[0123] To confirm the function of the gene products and directly establish their corresponding roles in NAA 10 biosynthesis, the inventors constructed a series of plasmids carrying various combinations of these genes and then transformed them into suitable *P. paxilli* hosts (Table 7) for heterologous production of NAA 10 precursors. Therefore, the CDS of the genes of interest from *H. pulicicidum* were amplified (see primers in Table 8) and cloned into the MIDAS 1 final vector pML1 (Table 9). At MIDAS 2, the cloned CDS was placed under the control of a heterologous promoter (ProUTR) and transcription terminator (UTRterm) module to produce a full-length TU (Table 10), which was then used to generate multi-gene plasmids (Table 11). The inventors used a set of *P. paxilli* knockout strains (Table 7) to perform functional complementation and pathway reconstruction to determine the function of genes in the NOD cluster. After transforming *Penicillium spp.* var. *paxilli* with a multigene plasmid, the inventors first determined the chemical phenotype of the transformant by normal-phase thin-layer chromatography (TLC, results not shown) of the fungal extract, followed by analysis by reversed-phase liquid chromatography-mass spectrometry (LC-MS). As determined by high-resolution mass spectrometry (HRMS), the inventors purified the newly expressed metabolite by semi-preparative reversed-phase high-performance liquid chromatography (HPLC) and performed nuclear magnetic resonance (NMR) spectroscopy analysis on the compound. 1 H. 13 C and HSQC, HMBC, COSY) are used for final identification.

[0124] Using this method, the inventors identified nodC (cDNA (SEQ ID NO: 23) and genomic DNA (SEQ ID NO: 22)) as a functional ortholog of paxC (cDNA (SEQ ID NO: 43) and genomic DNA (SEQ ID NO: 42)), and that NodC (SEQ ID NO: 24) mediates the production of GGI2 in H. pulicicidum, which is the second step in IDT biosynthesis. Figure 3 , trace ib, Figure 4NodC (SEQ ID NO: 24) shares 52.3% amino acid sequence identity with PaxC (SEQ ID NO: 44) from *Penicillium muscarinii* (Table 2). The inventors also identified nodM (cDNA (SEQ ID NO: 11) and genomic DNA (SEQ ID NO: 10)) as homologs of paxM (cDNA (SEQ ID NO: 46) and genomic DNA (SEQ ID NO: 45)), and NodM (SEQ ID NO: 12) as a GGI 2 monocyclic oxygenase that catalyzes the production of GGI 3a via monocyclic oxidation. Figure 3 Traces ii.b Figure 5 NodM (SEQ ID NO: 12) shares 48.6% sequence identity with PaxM (SEQ ID NO: 47) from *Penicillium muscarinii* (Table 3). Notably, the inventors show that NodM (SEQ ID NO: 12) is specifically a monocyclic oxygenase, unlike PaxM (SEQ ID NO: 47), which is a monocyclic or dicyclic oxygenase. The inventors also demonstrated that NodB (SEQ ID NO: 15) from *H. pulicicidum* acts as an IDT cyclase, which cyclizes the monocyclic oxidized GGI product 3a to form emindole SB 4a. Figure 3 Trajectory iii.b Figure 6 Furthermore, nodB (cDNA (SEQ ID NO: 14) and genomic DNA (SEQ ID NO: 13)) is a functional ortholog of paxB (cDNA (SEQ ID NO: 52) and genomic DNA (SEQ ID NO: 51)) from *Penicillium fungi*. Figure 3 Trajectory iv.b Figure 7 NodB (SEQ ID NO: 15) from H. pulicicidum shares 63% identity with PaxB (SEQ ID NO: 53) from P. paxilli (Table 4).

[0125] The dedicated NAA 10 core is NAF 5a, which is produced by the oxidation of the terminal methyl carbon C5” of emindole SB 4a. Figure 8B). Therefore, the inventors achieved NAF 5a production by co-expressing nodW (cDNA (SEQ ID NO: 2) and genomic DNA (SEQ ID NO: 1)) and nodM (cDNA (SEQ ID NO: 11) and genomic DNA (SEQ ID NO: 10)) in a paxM (cDNA (SEQ ID NO: 46) and genomic DNA (SEQ ID NO: 45)) deletion background (PN2257). Figure 3 trace vb Figure 9 This confirmed that the oxidase is a P450 oxygenase encoded by H. pulicicidum nodW (cDNA (SEQ ID NO: 2) and genomic DNA (SEQ ID NO: 1)).

[0126] To confirm that only five genes are essential for NAF 5a production in *Penicillium paxilli*, and to establish that other *Penicillium paxilli* IDT genes from the PAX cluster, absent in the paxM KO strain (PN2257), contribute to NAF5a production, the inventors assembled a multigene construct comprising paxG (genomic DNA (SEQ ID NO: 40)) from *Penicillium paxilli* and nodC (genomic DNA (SEQ ID NO: 22)), nodM (genomic DNA (SEQ ID NO: 10)), nodB (genomic DNA (SEQ ID NO: 13)), and nodW (genomic DNA (SEQ ID NO: 1)) from *H. pulicicidum*. This multigene construct was transformed into a *Penicillium paxilli* PAX cluster knockout strain (PN2250). As expected, expression of the five genes showed that NAF 5a was generated. Figure 10 ), and indicates that these 5 genes are in fact required for the biosynthesis of NAF 5a.

[0127] Building upon the work described herein, the inventors disclose the first five steps of using heterologous expression to identify the delivery of the NAA 10 core compound NAF 5a. Specifically, the inventors confirmed the function of four previously unknown genes from *H. pulicicidum*: nodC (cDNA (SEQ ID NO: 23) and genomic DNA (SEQ ID NO: 22)), nodM (cDNA (SEQ ID NO: 11) and genomic DNA (SEQ ID NO: 10)), nodB (cDNA (SEQ ID NO: 14) and genomic DNA (SEQ ID NO: 13)), and nodW (cDNA (SEQ ID NO: 2) and genomic DNA (SEQ ID NO: 1)), and discovered a second filamentous fungal species, *H. pulicicidum*, that appears to lack the secondary metabolic GGPPS gene but still produces IDT. Not wishing to be limited by theory, the inventors believe that *H. pulicicidum* relies on its primary metabolic GGPPS to provide GGPP for the IDT system. The absence of secondary metabolic GGPPS could explain why *H. pulicicidum* produces such small amounts of NA. The low amount of NA produced by *H. pulicicidum* poses a challenge for both elucidating the details of biosynthesis and the use of the compound or its derivatives. The inventors overcame both problems by using the efficient genome assembly of MIDAS in heterologous expression in *P. paxilli*. Furthermore, the inventors demonstrated that *P. paxilli*, due to its far more favorable growth conditions, is a suitable host for heterologous expression studies, enabling the inventors to confirm gene function faster and more easily than could be achieved relying on the biosynthetic machinery of *H. pulicicidum*.

[0128] The elucidation of the biosynthetic pathway for the heterologous production of NAF 5a in *Penicillium musculosus* (P. paxilli) provides a reasonable expectation of success for the gene product from *H. pulicicidum* responsible for the “decorative” step leading to the fully functionalized NAF 10. This reasonable expectation stems from the inventors’ identification of the nucleic acid CDS of the enzymes expected to encode isoprenylation to form polysporic acid E6a and oxidation to form polysporic acid D7a. Each of these nucleic acid sequences has been inferred and identified from the *H. pulicicidum* biosynthetic gene cluster described herein. In summary, the inventors’ work described herein demonstrates that heterologous expression of the IDT gene in a heterologous host is a viable method for producing natural products that are otherwise difficult to obtain and may be useful in many industrial sectors.

[0129] peptides

[0130] Therefore, in one aspect, the present invention relates to an isolated polypeptide comprising an amino acid sequence selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof.

[0131] Preferably, the functional variant or fragment has at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 99% amino acid sequence identity with the amino acid sequence selected from SEQ ID NO: NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56).

[0132] Preferably, the isolated polypeptide containing NodW (SEQ ID NO: 3) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0133] Preferably, the isolated polypeptide containing NodR (SEQ ID NO: 6) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0134] Preferably, the isolated polypeptide containing NodX (SEQ ID NO: 9) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0135] Preferably, the isolated polypeptide containing NodM (SEQ ID NO: 12) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0136] Preferably, the isolated polypeptide containing NodB (SEQ ID NO: 15) or a functional variant or fragment thereof has cyclase activity, preferably IDT cyclase activity.

[0137] Preferably, the isolated polypeptide containing NodO (SEQ ID NO: 18) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0138] Preferably, the isolated polypeptide containing NodJ (SEQ ID NO: 21) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0139] Preferably, the isolated polypeptide containing NodC (SEQ ID NO: 24) or a functional variant or fragment thereof has transferase activity, preferably GGT activity.

[0140] Preferably, the isolated polypeptide containing NodY1 (SEQ ID NO: 27) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0141] Preferably, the isolated polypeptide containing NodD2 (SEQ ID NO: 30) or a functional variant or fragment thereof has transferase activity, preferably isoprenyl transferase activity.

[0142] Preferably, the isolated polypeptide containing NodD1 (SEQ ID NO: 33) or a functional variant or fragment thereof has transferase activity, preferably isoprenyl transferase activity.

[0143] Preferably, the isolated polypeptide containing NodY2 (SEQ ID NO: 36) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0144] Preferably, the isolated polypeptide containing NodZ (SEQ ID NO: 39) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0145] In one embodiment, the isolated polypeptide comprises NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or functional variants or fragments thereof.

[0146] In one embodiment, the isolated polypeptide is substantially composed of NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or functional variants or fragments thereof.

[0147] In one embodiment, the isolated polypeptide is composed of SEQ ID NO: NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof.

[0148] polynucleotides

[0149] On the other hand, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NO: NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof.

[0150] Preferably, the functional variant or fragment has at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity with the amino acid sequence selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof.

[0151] Preferably, the isolated polynucleotide encodes a polypeptide containing NodW (SEQ ID NO: 3) or a functional variant or fragment thereof, which has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0152] Preferably, the isolated polynucleotide encodes a polypeptide containing NodR (SEQ ID NO: 6) or a functional variant or fragment thereof, which has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0153] Preferably, the isolated polynucleotide encodes a polypeptide containing NodX (SEQ ID NO: 9) or a functional variant or fragment thereof, which has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0154] Preferably, the isolated polynucleotide encodes a polypeptide containing NodM (SEQ ID NO: 12) or a functional variant or fragment thereof, which has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0155] Preferably, the isolated polynucleotide encodes a polypeptide containing NodB (SEQ ID NO: 15) or a functional variant or fragment thereof, which has cyclase activity, preferably IDT cyclase activity.

[0156] Preferably, the isolated polynucleotide encodes a polypeptide containing NodO (SEQ ID NO: 18) or a functional variant or fragment thereof, which has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0157] Preferably, the isolated polynucleotide encodes a polypeptide containing NodJ (SEQ ID NO: 21) or a functional variant or fragment thereof, which has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0158] Preferably, the isolated polynucleotide encodes a polypeptide containing NodC (SEQ ID NO: 24) or a functional variant or fragment thereof, which has transferase activity, preferably GGT activity.

[0159] Preferably, the isolated polynucleotide encodes a polypeptide containing NodY1 (SEQ ID NO: 27) or a functional variant or fragment thereof, which has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0160] Preferably, the isolated polynucleotide encodes a polypeptide containing NodD2 (SEQ ID NO: 30) or a functional variant or fragment thereof, which has transferase activity, preferably isoprenyl transferase activity.

[0161] Preferably, the isolated polynucleotide encodes a polypeptide containing NodD1 (SEQ ID NO: 33) or a functional variant or fragment thereof, which has transferase activity, preferably isoprenyl transferase activity.

[0162] Preferably, the isolated polynucleotide encodes a polypeptide containing NodY2 (SEQ ID NO: 36) or a functional variant or fragment thereof, which has oxygenase activity, preferably FAD-dependent oxygenase activity.

[0163] Preferably, the isolated polynucleotide encodes a polypeptide containing NodZ (SEQ ID NO: 39) or a functional variant or fragment thereof, which has oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0164] In one embodiment, the isolated polynucleotide encodes a polypeptide or a functional variant or fragment thereof comprising NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56).

[0165] In one embodiment, the isolated polynucleotide-encoded polypeptide is substantially composed of NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56) or functional variants or fragments thereof.

[0166] In one embodiment, the isolated polynucleotide-encoded polypeptide is composed of NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or functional variants or fragments thereof.

[0167] On the other hand, the present invention relates to an isolated polynucleotide, which is selected from nodW cDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), and nodD2. The nucleic acid sequences of the cDNA (SEQ ID NO: 29), nodD2 genomic DNA (SEQ ID NO: 28), nodD1 cDNA (SEQ ID NO: 32), nodD1 genomic DNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 genomic DNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ genomic DNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS genomic DNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI genomic DNA (SEQ ID NO: 54) have at least 70% nucleic acid sequence identity.

[0168] Preferably, the isolated polynucleotide is selected from nodW cDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), and nodD2. The nucleic acid sequences of the cDNA (SEQ ID NO: 29), nodD2 genomic DNA (SEQ ID NO: 28), nodD1 cDNA (SEQ ID NO: 32), nodD1 genomic DNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 genomic DNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ genomic DNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS genomic DNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI genomic DNA (SEQ ID NO: 54) have a nucleic acid sequence identity of at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 99%.

[0169] In one embodiment, the isolated polynucleotide comprises a subset selected from nodW cDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), and nodD2. Nucleic acid sequences of cDNA (SEQ ID NO: 29), nodD2 genomic DNA (SEQ ID NO: 28), nodD1 cDNA (SEQ ID NO: 32), nodD1 genomic DNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 genomic DNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ genomic DNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS genomic DNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI genomic DNA (SEQ ID NO: 54).

[0170] In one embodiment, the isolated polynucleotide is substantially selected from nodW cDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodX cDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), and nodD2. The nucleic acid sequences of the following cDNAs are: nodD2 cDNA (SEQ ID NO: 29), nodD1 cDNA (SEQ ID NO: 32), nodD1 cDNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 cDNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ cDNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS cDNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI cDNA (SEQ ID NO: 54).

[0171] In one embodiment, the isolated polynucleotide is selected from nodWcDNA (SEQ ID NO: 2), nodW genomic DNA (SEQ ID NO: 1), nodR cDNA (SEQ ID NO: 5), nodR genomic DNA (SEQ ID NO: 4), nodXcDNA (SEQ ID NO: 8), nodX genomic DNA (SEQ ID NO: 7), nodM cDNA (SEQ ID NO: 11), nodM genomic DNA (SEQ ID NO: 10), nodB cDNA (SEQ ID NO: 14), nodB genomic DNA (SEQ ID NO: 13), nodOcDNA (SEQ ID NO: 17), nodO genomic DNA (SEQ ID NO: 16), nodJ cDNA (SEQ ID NO: 20), nodJ genomic DNA (SEQ ID NO: 19), nodC cDNA (SEQ ID NO: 23), nodC genomic DNA (SEQ ID NO: 22), nodY1 cDNA (SEQ ID NO: 26), nodY1 genomic DNA (SEQ ID NO: 25), nodD2 The nucleic acid sequences of the following cDNAs are: nodD2 cDNA (SEQ ID NO: 29), nodD1 cDNA (SEQ ID NO: 32), nodD1 cDNA (SEQ ID NO: 31), nodY2 cDNA (SEQ ID NO: 35), nodY2 cDNA (SEQ ID NO: 34), nodZ cDNA (SEQ ID NO: 38), nodZ cDNA (SEQ ID NO: 37), nodS cDNA (SEQ ID NO: 49), nodS cDNA (SEQ ID NO: 48), nodI cDNA (SEQ ID NO: 55), and nodI cDNA (SEQ ID NO: 54).

[0172] The nucleic acid molecules of this invention or otherwise described herein are preferably isolated. They can be isolated from biological samples using a variety of different techniques known to those skilled in the art. For example, these polynucleotides can be isolated using polymerase chain reaction (PCR) as known in the art. The nucleic acid molecules of this invention can be amplified using primers derived from the polynucleotide sequences of this invention as defined herein.

[0173] Other methods for isolating polynucleotides include using all or some of the polynucleotides of the present invention as hybridization probes. Techniques for hybridizing labeled polynucleotide probes to polynucleotides immobilized on a solid support, such as a nitrocellulose filter membrane or a nylon membrane, can be used to screen genomic or cDNA libraries. Similarly, probes can be coupled to beads and hybridized to target sequences. Separation can be achieved using known techniques such as magnetic separation. Hybridization and washing conditions suitable for stringency are believed to be within the scope of the art.

[0174] Polynucleotide fragments can be produced using techniques known in the art, such as restriction endonuclease digestion and oligonucleotide synthesis.

[0175] In methods known in the art, partial polynucleotide sequences can be used as probes to identify corresponding full-length polynucleotide sequences in a sample. These methods include PCR-based methods, 5'RACE and hybridization-based methods, and computer / database-based methods known in the art. Detectable markers such as radioisotopes, fluorescence, chemiluminescence, and bioluminescence markers can be used to facilitate detection. Reverse PCR also allows the acquisition of unknown sequences flanking the polynucleotide sequences disclosed herein, starting with primers based on known regions, as is known and used in the art. The methods use several restriction enzymes to generate suitable fragments in known regions of the gene. These fragments are then circularized via intramolecular linking and used as PCR templates. Divergent primers are designed from the known regions. For the physical assembly of full-length clones, standard molecular biology methods can be utilized as known in the art. Primers and primer pairs that allow the amplification of the polynucleotides of the present invention also form another aspect of the invention.

[0176] Variant polynucleotides (including orthologs) can be identified using the methods described. Variant polynucleotides can be identified using PCR-based methods known in the art. Typically, the polynucleotide sequence of primers that can be used to amplify variant polynucleotide molecules by PCR can be based on the sequence encoding a conserved region of the corresponding amino acid sequence.

[0177] Other methods for identifying variant polynucleotides include screening genomic or cDNA libraries using all or part of a specified polynucleotide as a hybridization probe, as described above. Typically, probes based on sequences encoding conserved regions of the corresponding amino acid sequence can be used. Hybridization conditions can also be less stringent than those used when screening for sequences consistent with the probe.

[0178] On the other hand, the present invention relates to a TU comprising at least one isolated polynucleotide described herein. In one embodiment, the TU is contained in a vector, preferably an expression vector. In one embodiment, the vector is selected from plasmids, BACs, PACs, YACs, phages, phage particles, and granules. Preferably, the vector is a plasmid.

[0179] On the other hand, the present invention relates to a carrier that encodes an isolated polypeptide or a functional variant or fragment thereof according to the present invention.

[0180] On the other hand, the present invention relates to a vector comprising the isolated nucleic acid sequence according to the present invention.

[0181] In one embodiment, the isolated nucleic acid sequence is contained in a TU.

[0182] In one embodiment, the vector is selected from plasmids, BACs, PACs, YACs, bacteriophages, phage particles, and granules. Preferably, the vector is a plasmid. In one embodiment, the vector is an expression vector.

[0183] The TU containing the polynucleotide of the present invention can be incorporated into any suitable vector capable of expressing the polynucleotide in vitro or in a host cell, or, where applicable, the encoding polypeptide of the present invention. Preferably, the vector is an expression vector. Examples of suitable expression vectors include, but are not limited to, plasmid DNA vectors, viral DNA vectors (e.g., adenovirus and adeno-associated virus), or viral RNA vectors (e.g., retroviral vectors). In some embodiments, the plasmid and / or phage vector may be selected from the following vectors or variants thereof, including pUC18, pU19, Mp18, Mp19, ColE1, PCR1 and pKRC, λgt10 and M13 plasmids such as pBR322, pACYC184, pT127, RP4, p1J101, SV40 and BPV. Vectors such as, but not limited to, entrapments, YAC, BAC shuttle vectors such as pSA3, PAT28 transposons (e.g., described in US 5,792,294), etc., are also included.

[0184] Suitable viral vectors include, but are not limited to, vectors derived from adenoviruses (AV), adeno-associated viruses (AAV), retroviruses (e.g., lentiviruses (LV), rhabdoviruses, murine leukemia viruses), herpesviruses, etc. The viral vectors used herein can be suitably modified by using pseudo-packaging with envelope proteins or other surface antigens derived from other viruses, or by replacing different viral capsid proteins, as is known and used in the art.

[0185] In one embodiment, the expression vector comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten isolated polynucleotides described herein.

[0186] In one embodiment, the expression vector comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten TUs described herein.

[0187] In one embodiment, the vector is a component of the cloning system. In one embodiment, the cloning system is useful for manufacturing gene constructs containing at least one TU.

[0188] In one embodiment, the vector is included in a vector group, which is part of a cloning system. In one embodiment, the cloning system is useful for manufacturing gene constructs containing at least one TU.

[0189] In one embodiment, the cloning system is useful for manufacturing genetic constructs containing at least one TU.

[0190] In one embodiment, the gene construct is a multi-gene construct containing at least two TUs. In another embodiment, the multi-gene construct contains at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, or preferably at least ten TUs.

[0191] The TUs described herein may comprise one or more of the disclosed polynucleotide sequences of this invention and / or polynucleotides encoding the disclosed polypeptides. The TUs may be constructed to drive the expression of at least one polypeptide involved in the biosynthesis of NAA10, either in vitro or in vivo. In one embodiment, the TU comprises a polynucleotide of this invention operably linked to a 5′ or 3′ untranslated regulatory sequence. The design of a particular TU depends on a variety of factors, including the host cell in which the operably linked polynucleotide will be expressed and the desired polynucleotide expression level.

[0192] Similarly, for a TU, the selection of various promoters, enhancers, and / or other genetic elements depends on a variety of factors, including the host cell and expression level discussed above. In one embodiment, the TU comprises a homologous promoter operably linked to the polynucleotide of the present invention. In another embodiment, the expression cassette comprises a heterologous promoter operably linked to the polynucleotide of the present invention. In one embodiment, the homologous or heterologous promoter is an inducible, repressible, or regulated promoter. Suitable promoters can be selected and used under suitable conditions to guide high-level expression of the polynucleotide of the present invention. Many such elements are described in the literature and are available from commercial suppliers.

[0193] By way of example only, the promoter useful in the expression cassette can be any suitable eukaryotic or prokaryotic promoter. In one embodiment, the eukaryotic promoter can be eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). The expression level of the operable polynucleotide in a particular cell type will be determined by the presence (or absence) of a specific gene regulatory sequence (e.g., enhancer, silencer, etc.) in the vicinity. Any suitable promoter / enhancer combination (see the eukaryotic promoter database EPDB) can be used to drive the expression of the polynucleotide of the present invention.

[0194] Other promoters useful in expression cassettes include β-lactamases, alkaline phosphatases, tryptophan, and the tac promoter system, which are well known in the art. Yeast promoters include, but are not limited to, 3-phosphoglycerate kinase, enolase, hexokinase, pyruvate decarboxylase, glucokinase, and glyceraldehyde-3-phosphate dehydrogenase.

[0195] Useful prokaryotic promoters in expression cassettes include constitutive promoters known in the art (e.g., the int promoter of phage λ and the bla promoter of the β-lactamase gene sequence of pBR322) and regulated promoters (e.g., lacZ, recA, and gal). Expression may also require a ribosome binding site upstream of the CDS.

[0196] Useful enhancers in TU include the SV40 enhancer, the early promoter enhancer of cytomegalovirus, globin, albumin, insulin, etc.

[0197] In one implementation, the TU can be driven by a T3, T7, or SP6 cytoplasmic expression system.

[0198] The selection of a specific promoter / enhancer / cell type combination for protein expression is within the general techniques of those in the field of molecular biology (see, for example, Sambrook et al., (1989), which is incorporated herein by reference).

[0199] On the other hand, the present invention relates to an isolated host cell comprising isolated polypeptides, isolated polynucleotides, TUs and / or isolated vectors according to the present invention.

[0200] In one embodiment, the isolated host cell is a prokaryotic or eukaryotic cell. The most commonly used prokaryotic organism as a host cell is a strain of *Escherichia coli*. Other prokaryotic hosts include, but are not limited to, *Pseudomonas*, *Bacillus*, *Serratia*, *Klebsiella*, *Streptomyces*, *Listeria*, *Salmonella*, and mycobacteria.

[0201] In one embodiment, the eukaryotic cell is an animal cell, plant cell, fungal cell, or protist cell. In one embodiment, the animal cell is an insect cell or mammalian cell. In one embodiment, the fungal cell is a single cell of a unicellular fungal host strain. In one embodiment, the fungal cell comprises fungal hyphae or mycelium of a fungal host strain.

[0202] In one embodiment, the fungal cells, hyphae, or mycelia of the fungal host strain are derived from *Aspergillus*, *Trichoderma*, *Neurospora*, *Fusarium*, *Morchella*, *Aureobasidium*, *Candida*, *Geotrichum*, *Yersinia*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Aspergillus*, *Hansenula*, *Pichia pastoris*, *Kluyveromyces*, *Schizosacchari*, *Monascus*, *Morchella*, *Cryptonectria*, *Enteromorpha*, *Cryptospira*, *Hypericum*, *Pichia pastoris*, *Fusarium*, *Schizophyllum*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, *Pleurotus*, *Penicillium*, *Lentinula*, *Lentinula*, *Auricularia*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, *Rhizopus*, or *Saccharomyces* genus, preferably *Penicillium*, *Aspergillus*, *Saccharomyces*, *Pichia pastoris*, *Pseudomonas*, and *Pseudomonas* genus. Preferably, the fungal cells are derived from *Saccharomyces* genus. Preferably, the fungal hyphae or mycelia are derived from *Penicillium* genus, preferably *Penicillium paxilli*.

[0203] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising heterologously expressing at least one polypeptide, isolated nucleic acid sequence, TU or vector according to the present invention in isolated host cells.

[0204] In one implementation, the NA is selected from... Figure 1 The NA is a set of NAs described in the diagram. Preferably, the NA is NAF 5a or NAA 10, more preferably NAA 10.

[0205] In one embodiment, the polypeptide is a polypeptide or a functional variant or fragment according to the present invention.

[0206] As embodiments within this aspect of the invention, various different embodiments are specifically envisioned herein with respect to any other aspect of the invention relating to heterologous expression (including the selection of suitable regulatory sequences), expression cassettes, genetic elements, TUs, multigene constructs, host cells, and vectors.

[0207] In a particular embodiment, heterologous expression of the polypeptide comprises expressing at least one polynucleotide according to the invention or at least one TU encoding at least one polypeptide of the invention from at least one vector described herein in the mycelium of an isolated fungal host cell or an isolated fungal strain described herein. In one embodiment, the polypeptide is NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), or NodZ (SEQ ID NO: 39), preferably the polypeptide is an enzyme catalyzing the biotransformation from NAB 9 to NAA 10. In one embodiment, the fungal cell or strain is a Penicillium cell or strain, preferably *Penicillium fungi* (P. paxilli).

[0208] In one embodiment, the TU is included in a multigene construct containing at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, and / or at least ten polynucleotides encoding polypeptides according to the present invention.

[0209] On the other hand, the present invention relates to at least one nano-acid (NA) manufactured by the method of the present invention. In one embodiment, the NA is selected from... Figure 1 The NA is depicted in the diagram. Preferably, the NA is NAF 5a or NAA 10, more preferably NAA 10.

[0210] On the other hand, the present invention relates to an isolated polypeptide or a functional variant or fragment thereof derived from Hypoxylon spp., which catalyzes a biochemical reaction in the biosynthetic pathway from GGI 2 to NAA 10.

[0211] On the other hand, the present invention relates to an isolated polynucleotide encoding at least one polypeptide derived from Hypoxylon spp., said polypeptide catalyzing a biochemical reaction in the biosynthetic pathway that produces NAA 10 from GGI 2.

[0212] In one embodiment, the isolated polypeptide is an oxygenase, preferably a cytochrome P450 oxygenase or a FAD-dependent oxygenase. Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodJ (SEQ ID NO: 21), or NodZ (SEQ ID NO: 39). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12), NodO (SEQ ID NO: 18), NodY1 (SEQ ID NO: 27), or NodY2 (SEQ ID NO: 36). In one embodiment, the isolated polypeptide is a transferase, preferably GGT or an isoprenyltransferase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the isoprenyltransferase is NodD1 (SEQ ID NO: 33) or NodD2 (SEQ ID NO: 30). In one embodiment, the isolated polypeptide is an IDT cyclase. Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). In one embodiment, the isolated polypeptide is NodS (SEQ ID NO: 50). In one embodiment, the isolated polypeptide is NodI (SEQ ID NO: 56).

[0213] In one embodiment, the isolated polypeptide catalyzes a biochemical reaction in the biosynthetic pathway from GGI 2 to NAF 5a. Preferably, the isolated polypeptide is GGT, a FAD-dependent oxygenase, an IDT cyclase, or a cytochrome P450 oxygenase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12). Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3).

[0214] In one embodiment, the isolated polypeptide or a functional variant or fragment thereof is encoded by a nucleic acid according to the present invention.

[0215] On the other hand, the present invention relates to a method for manufacturing at least one Hypoxylon spp. polypeptide or a functional variant or fragment thereof, the method comprising heterologously expressing an isolated nucleic acid sequence, TU or vector according to the present invention in isolated host cells.

[0216] In one embodiment, the at least one Hypoxylon spp. polypeptide is a polypeptide contemplated herein for any other aspect of the invention.

[0217] In one embodiment, the at least one Hypoxylon spp. polypeptide is a polypeptide comprising the amino acid sequence of NodW (SEQ ID NO: 3) or a functional variant or fragment thereof. Preferably, the polypeptide is substantially composed of or constitutes NodW (SEQ ID NO: 3). In one embodiment, the isolated host cell comprises fungal mycelium of the genus Penicillium, preferably Penicillium paxilli.

[0218] Various different embodiments of the invention are specifically envisioned for this aspect of the invention, which relate to heterologous expression (including the selection of suitable regulatory sequences), genetic elements, TUs, multigene constructs, host cells, and vectors, as well as any other aspect thereof.

[0219] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising heterologously expressing at least one polypeptide in isolated host cells that catalyzes a biochemical reaction in the biosynthetic pathway that produces NAA10 from GGI2.

[0220] In one embodiment, the at least one polypeptide is an oxygenase, preferably a cytochrome P450 oxygenase or a FAD-dependent oxygenase. Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodJ (SEQ ID NO: 21), or NodZ (SEQ ID NO: 39). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12), NodO (SEQ ID NO: 18), NodY1 (SEQ ID NO: 27), or NodY2 (SEQ ID NO: 36). In one embodiment, the isolated polypeptide is a transferase, preferably GGT or an isoprenyltransferase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the isoprenyltransferase is NodD1 (SEQ ID NO: 33) or NodD2 (SEQ ID NO: 30). In one embodiment, the isolated polypeptide is an IDT cyclase. Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). In one embodiment, the isolated polypeptide is NodS (SEQ ID NO: 50). In one embodiment, the isolated polypeptide is NodI (SEQ ID NO: 56).

[0221] In one embodiment, the at least one polypeptide catalyzes a biochemical reaction in the biosynthetic pathway from GGI 2 to NAF 5a. Preferably, the at least one polypeptide is GGT, a FAD-dependent oxygenase, an IDT cyclase, or a cytochrome P450 oxygenase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12). Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3).

[0222] In one embodiment, the at least one polypeptide comprises the amino acid sequence of NodW (SEQ ID NO: 3) or a functional variant or fragment thereof. Preferably, the polypeptide is substantially composed of or constitutes NodW (SEQ ID NO: 3). In one embodiment, the isolated host cell comprises fungal mycelium of Penicillium, preferably Penicillium paxilli.

[0223] Various different embodiments of the invention are specifically envisioned for this aspect of the invention, which relate to heterologous expression (including the selection of suitable regulatory sequences), genetic elements, TUs, multigene constructs, host cells, and vectors, as well as any other aspect thereof.

[0224] On the other hand, the present invention relates to an isolated host cell that expresses at least one heterologous polypeptide that catalyzes the conversion of substrates in a biosynthetic pathway leading to the formation of NAA10.

[0225] In one embodiment, at least one heterologous polypeptide catalyzes the conversion of a substrate in a biosynthetic pathway leading to the formation of NAF5a.

[0226] In one embodiment, the substrate is selected from GGPP1a, indole-3-glycerophosphate 1b, GGI2, monoepoxide GGI3a, emindole SB4a, NAF5a, NAE6a, NAD7a, NAC8, and NAB9.

[0227] In one embodiment, the transformation is selected from condensation, oxidation, or cyclization.

[0228] In one embodiment, the substrates to be converted are GGPP 1a and indole-3-glycerophosphate 1b, and the conversion is a condensation.

[0229] In one embodiment, the substrate being converted is GGI 2, and the conversion is oxidation.

[0230] In one embodiment, the substrate being transformed is a monocyclic oxidized GGI 3a, and the transformation is cyclization.

[0231] In one embodiment, the substrate being transformed is emindole SB 4a, and the transformation is oxidation.

[0232] In one embodiment, the substrate being transformed is NAF 5a, and the transformation is a condensation.

[0233] In one embodiment, the substrate being transformed is NAE 6a, and the transformation is oxidation.

[0234] In one embodiment, the substrate being transformed is NAD 7a, and the transformation is oxidation and condensation.

[0235] In one embodiment, the substrate being converted is NAC 8, and the conversion is oxidation.

[0236] In one embodiment, the substrate being converted is NAB 9, and the conversion is oxidation.

[0237] On the other hand, the present invention relates to an isolated host cell that produces at least one polypeptide involved in the biosynthetic pathway that generates NAA10 through heterologous expression.

[0238] In one embodiment, the at least one polypeptide catalyzes a biochemical reaction in the biosynthetic pathway from GGI 2 to NAF 5a. Preferably, the at least one polypeptide is GGT, a FAD-dependent oxygenase, an IDT cyclase, or a cytochrome P450 oxygenase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12). Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3).

[0239] In certain embodiments specifically conceived for this aspect of the invention, the at least one polypeptide is a polypeptide involved in the biosynthetic pathway for the production of NAA10 as defined herein for any other aspect of the invention.

[0240] In one embodiment, at least one polypeptide is a polypeptide of the present invention or a functional variant or fragment thereof. In one embodiment, the polypeptide or a functional variant or fragment thereof is encoded by a nucleic acid sequence of the present invention.

[0241] In one embodiment, the at least one polypeptide participates in the biosynthetic pathway that produces NAF 5a. In one embodiment, the at least one polypeptide comprises the amino acid sequence of NodW (SEQ ID NO: 3) or a functional variant or fragment thereof. Preferably, the polypeptide is substantially composed of or constitutes NodW (SEQ ID NO: 3). In one embodiment, the isolated host cell comprises penicillin, preferably fungal mycelium of *Penicillium muscarinii* (P. paxilli).

[0242] Various different embodiments of the invention are specifically envisioned for this aspect of the invention, which relate to heterologous expression (including the selection of suitable regulatory sequences), genetic elements, TUs, multigene constructs, host cells, and vectors, as well as any other aspect thereof.

[0243] On the other hand, the present invention relates to a method for producing at least one NA, the method comprising contacting a substrate comprising carbohydrates with recombinant cells transformed with nucleic acids, the transformation resulting in an increased level or activity of a polypeptide selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof, compared with the cells before transformation, such that the substrate is metabolized into at least one NA.

[0244] In one embodiment, the nucleic acid encodes at least one polypeptide that catalyzes a biochemical reaction in the biosynthetic pathway from GGI 2 to NAF 5a, preferably catalyzing a biochemical reaction from emindole SB4a to NAF 5a.

[0245] In one embodiment, the recombinant host cell is an isolated host cell of the present invention as described herein.

[0246] In one embodiment, the sugars are contained in a culture medium. In another embodiment, the culture medium is CDYE or a variant thereof that supports the growth of the recombinant cells.

[0247] In one embodiment, the nucleic acid encodes at least one polypeptide, which is an oxygenase, preferably a cytochrome P450 oxygenase or a FAD-dependent oxygenase. Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodJ (SEQ ID NO: 21), or NodZ (SEQ ID NO: 39). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12), NodO (SEQ ID NO: 18), NodY1 (SEQ ID NO: 27), or NodY2 (SEQ ID NO: 36). In one embodiment, the isolated polypeptide is a transferase, preferably GGT or an isoprenyltransferase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the isoprenyltransferase is NodD1 (SEQ ID NO: 33) or NodD2 (SEQ ID NO: 30). In one embodiment, the isolated polypeptide is an IDT cyclase. Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). In one embodiment, the isolated polypeptide is NodS (SEQ ID NO: 50). In one embodiment, the isolated polypeptide is NodI (SEQ ID NO: 56).

[0248] In one embodiment, the nucleic acid encodes at least one GGT, FAD-dependent oxygenase, IDT cyclase, or cytochrome P450 oxygenase. In one embodiment, the nucleic acid encodes at least two, preferably at least three, and preferably at least four of GGT, FAD-dependent oxygenase, IDT cyclase, or cytochrome P450 oxygenase. Preferably, the GGT is NodC (SEQ ID NO: 24). Preferably, the FAD-dependent oxygenase is NodM (SEQ ID NO: 12). Preferably, the IDT cyclase is NodB (SEQ ID NO: 15). Preferably, the cytochrome P450 oxygenase is NodW (SEQ ID NO: 3).

[0249] In one embodiment, a polypeptide selected from NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), NodO (SEQ ID NO: 18), NodJ (SEQ ID NO: 21), NodC (SEQ ID NO: 24), NodY1 (SEQ ID NO: 27), NodD2 (SEQ ID NO: 30), NodD1 (SEQ ID NO: 33), NodY2 (SEQ ID NO: 36), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or a functional variant or fragment thereof, comprising the NodW (SEQ ID NO: 3), NodR (SEQ ID NO: 6), NodX (SEQ ID NO: 9), NodM (SEQ ID NO: 12), NodB (SEQ ID NO: 15), and NodO (SEQ ID NO: 56) of the present invention, is included. The amino acid sequences of NodJ (SEQ ID NO: 18), NodC (SEQ ID NO: 21), NodY1 (SEQ ID NO: 24), NodD2 (SEQ ID NO: 27), NodD1 (SEQ ID NO: 30), NodY2 (SEQ ID NO: 33), NodZ (SEQ ID NO: 39), NodS (SEQ ID NO: 50), and NodI (SEQ ID NO: 56), or their functional variants or fragments.

[0250] In one embodiment, the polypeptide comprises the amino acid sequence of NodW (SEQ ID NO: 3) or a functional variant or fragment thereof. Preferably, the polypeptide is substantially composed of or constitutes NodW (SEQ ID NO: 3). In one embodiment, the isolated host cell comprises penicillin, preferably fungal mycelium of Penicillium muscarinii (P. paxilli).

[0251] Various different embodiments of the invention are specifically envisioned for this aspect of the invention, which relate to heterologous expression (including the selection of suitable regulatory sequences), genetic elements, TUs, multigene constructs, host cells, and vectors, as well as any other aspect thereof.

[0252] In one embodiment, the at least one heterologous or introduced homologous nucleic acid sequence is at least one NAA10 biosynthetic gene selected from nodW, nodR, nodX, nodM, nodB, nodO, nodJ, nodC, nodY1, nodD2, nodD1, nodY2, nodZ, nodS, and nodI described herein.

[0253] In one embodiment, one of the two different GGPPS enzymes is produced by heterologous expression in H. pulicicidum.

[0254] In one embodiment, one of the two different GGPPS enzymes is encoded by a second copy of the original H. pulicicidum gene that encodes the GGPPS enzyme.

[0255] On the other hand, the present invention relates to an isolated strain of Hypoxylon pulicicidum containing genetic modifications that lead to increased biosynthesis of NAA10.

[0256] In one embodiment, the isolated strain contains increased expression of at least one GGPPS enzyme compared to a control strain of H. pulicicidum, preferably H. pulicicidum ATCC 74245.

[0257] In one embodiment, the increased expression is an enhanced expression of the primary GGPPS gene of H. pulicicidum induced by modification of genetic regulatory elements.

[0258] In one embodiment, modification of the genetic regulatory element includes operatively linking the original major GGPPS gene to an optional or modified promoter.

[0259] In one embodiment, the modification of the genetic regulatory element includes operatively linking the original major GGPPS gene to a more robust original promoter. In one embodiment, the original major GGPPS gene is an introduced homolog.

[0260] In one embodiment, the enhanced expression is the result of heterologous expression of a biosynthetic gene that contributes to NAA10 biosynthesis.

[0261] In one embodiment, the enhanced expression is caused by the expression of a heterologous gene in H. pulicicidum having equivalent biochemical function to a gene identified in the Nod cluster as described herein.

[0262] In one embodiment, the increased expression is caused by the expression of a heterologous gene in H. pulicicidum that addresses the supply limitations of substrate compounds or biosynthetic intermediates required for NAA 10 biosynthesis.

[0263] In one embodiment, the enhanced expression is caused by heterologous expression in H. pulicicidum of any gene encoding the GGT that catalyzes the condensation of GGPP 1a and indole-3-glycerophosphate 1b to produce 3-geranylgeranylindole 2.

[0264] In one implementation, the increased expression is caused by heterologous expression of any gene encoding GGPPS in H. pulicicidum.

[0265] In one embodiment, the enhanced expression is caused by heterologous expression in H. pulicicidum of any gene encoding a FAD-dependent oxidase that produces the monocyclic oxidation of GGI product 3a.

[0266] In one embodiment, the enhanced expression is caused by heterologous expression in H. pulicicidum of any gene encoding an enzyme that cyclizes the monocyclic oxidized GGI 3a to produce emindole SB 4a.

[0267] In one embodiment, the enhanced expression is caused by heterologous expression in H. pulicicidum of any gene encoding an enzyme that oxidizes emindole SB 4a to produce polysporic acid.

[0268] In one embodiment, the enhanced expression is caused by at least one genetic modification that results in enhanced expression of NA biosynthesis genes selected from nodW, nodR, nodX, nodM, nodB, nodO, nodJ, nodC, nodY1, nodD2, nodD1, nodY2, nodZ, nodS, and nodI as described herein.

[0269] On the other hand, the present invention relates to a method for manufacturing NAA10, the method comprising expressing at least one heterologous nucleic acid sequence in Hypoxylon pulicicidum, wherein the at least one heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway that produces NAA10.

[0270] Various different embodiments of the invention are specifically envisioned for this aspect of the invention relating to the isolated Hypoxylon pulicicidum strains described herein, including any other aspect relating to increased expression of NAA 10, and also including all embodiments described herein relating to heterologous expression (including the selection of suitable regulatory sequences), genetic elements, TUs, multigene constructs, host cells, and vectors.

[0271] In this specification, references to patent specifications, other external documents, or other sources of information are generally made for the purpose of providing background to the discussion of the features of the invention. Unless otherwise stated, references to such external documents should not be construed as an admission that such documents or sources are prior art or part of common general knowledge in the art within any jurisdiction.

[0272] The invention will now be described in a non-limiting manner with reference to the following embodiments. Example

[0273] Materials and methods

[0274] gDNA isolation for genome sequencing and TUM amplification

[0275] Genomic DNA used for PCR amplification of genome sequencing and TUM, following the method of Byrd et al. 26 Modifications were made to the Penicillium paxilli fungal material. 26601 TM (PN2013) 24 and Hypoxylon pulicicidum strain 74245 TM，25 Separation. Milli- was prepared in 125 mL conical shake flasks with 25 mL aliquots. Sterile 2.4% (w / v) Difco in water TM Potato Dextrose Broth (Becton, Dickinson and Company, Maryland, USA), and 5×10 6 One spore or ~1cm 2Inoculate freshly ground mycelium (for strains used for non-spore reproduction). Incubate the culture at 22°C with shaking (200 rpm) for 2–4 days. Filter the fermentation broth through a sterile diaper liner and wash the mycelium three times with sterile water. Transfer the mycelium to sterile 15 mL centrifuge tubes and freeze-dry in liquid nitrogen for 24–48 hours. Grind 15–20 mg of the freeze-dried mycelium into powder in a mortar containing liquid nitrogen. Transfer the ground mycelium to a 2 mL tube and resuspend it in 1 mL of extraction buffer (150 mM EDTA, 50 mM Tris-HCl, and 1% (w / v) sodium lauroyl sarcosinate). Add 1.6 mg of proteinase K to the tube and incubate the contents at 37°C for 30 min. Centrifuge the tube at 13,000 rpm for 10 min and transfer the supernatant to a new 2 mL tube. Add 500 μL of phenol and 500 μL of chloroform to the tube, vortex to mix the contents, and then centrifuge at 13,000 rpm for 10 min. Transfer the aqueous phase to a new 2 mL tube and wash twice with 500 μL of phenol and 500 μL of chloroform as described above. Then transfer the aqueous phase to a new 2 mL tube and wash with 1 mL of chloroform (vortex and centrifuge at 13,000 rpm for 10 min). Transfer the aqueous phase to a new 2 mL tube and mix with 1 mL of cold isopropanol. Precipitate the DNA overnight at -20 °C and centrifuge at 13,000 rpm for 10 min. Discard the supernatant and resuspend the DNA in 1 mL of 1 M NaCl. Incubate the tube at room temperature for 10 min, and then centrifuge at 13,000 rpm for 10 min to deposit the polysaccharide. Transfer the supernatant to a new tube and mix with 1 mL of isopropanol. Incubate the tube at room temperature for 10 min, and then centrifuge at 13,000 rpm for 10 min. Discard the supernatant and add 1 mL of cold 70% ethanol to the sediment without resuspending. Centrifuge the tube at 13,000 rpm for 2 min and discard the supernatant. Centrifuge the tube at 13,000 rpm for 1 min and aspirate the residual 70% ethanol. Vacuum dry the sediment at room temperature and resuspend it in 50 μL of Milli- It is stored in water at -20°C.

[0276] MIDAS Design Overview

[0277] MIDAS Toolkit is based on Kinmen assembly technology. 27MIDAS utilizes the ability of IIS-type restriction enzymes to seamlessly ligate multiple DNA fragments together in a single reaction. MIDAS employs three IIS-type restriction enzymes, AarI, BsaI, and BsmBI, which generate user-defined 4bp single-stranded overhangs after cleavage. By selecting suitable overhangs and aligning the IIS-type sites flanking each DNA fragment, multiple fragments can be assembled in an ordered (oriented) manner into a recipient plasmid (also known as the final vector) using a one-pot restriction-ligation reaction. The recipient plasmid contains marker genes (typically the lacZα gene for blue / white selection) with two divergent recognition sites flanking the IIS-type enzymes; these elements, collectively referred to as the "Golden Gate cloning box," are replaced by insert fragments during the assembly reaction.

[0278] Other recently described modular assembly technologies based on Kinmen 28-32 Similarly, the assembly of genes and multi-gene constructs using MIDAS is a hierarchical process. In the first level (MIDAS Level 1), functional modules (promoters, CDS, transcription terminators, tags, etc.) are cloned into the Level 1 final vector (pML1), where they form a library of reusable, sequence-verifiable components. The complementary design of the modules and the final vector ensures that after cloning into pML1, these modules can be released from the vector via digestion with BsaI.

[0279] In the second stage (stage 2), a BsaI-mediated Golden Gate reaction is used to release the compatible, group-verified stage 1 module from pML1 and assemble it into the stage 2 final vector (pML2), resulting in a stage 2 plasmid containing eukaryotic TUs. Similarly, design rules ensure that each assembled TU can be released from the pML2 vector, this time by digestion with AarI or BsmBI (depending on the pML2 vector in which the TU was assembled).

[0280] At level 3, using a 4arI or BsmBI-mediated Golden Gate reaction, the TUs assembled at level 2 are released from the pML2 plasmid and sequentially assembled together in the level 3 final vector (pML3) to form a functional multigene construct, which can then be transformed into the desired expression host.

[0281] Level 1: Module Cloning

[0282] At stage 1, functional TUM is produced as a PCR product or as a synthetic polynucleotide sequence (from a gene synthesis company) and cloned into the stage 1 final vector (pML1) via BsmBI-mediated Golden Gate cloning. For cloning into the pML1 vector, PCR primers are designed such that each amplified TUM is flanked by two converging BsmBI sites, BsmBI[CTCG] and [AGAC]BsmBI, whose sticky ends produced after restriction enzyme digestion are compatible with the sticky ends of the BsmBI sites present in the pML1 final vector. Therefore, the Golden Gate cloning cassette in pML1 consists of lacZα scoring markers with two diverging BsmBI sites flanking it: 5'-[CTCG]BsmBI-lacZα-BsmBI[AGAC]-3'( Figure 11 A).

[0283] To enable subsequent (i.e., grade 2) assembly of the full-length TU, each TUM was designed with four module-specific nucleotides (NNNN) flanking at the 5' end and four module-specific nucleotides (NNNN) flanking at the 3' end, which were included as part of the PCR primer sequence. The complementary design of the amplified modules and the pML1 vector ensured that when the amplified TUM was cloned into pML1 using a BsmBI-mediated gate reaction, each TUM became flanked by converging BsaI recognition sites. Furthermore, when the modules were released from pML1 during the subsequent (i.e., grade 2) BsaI-mediated gate assembly of the full-length TU, the module-specific nucleotides (NNNN and NNNN) became BsaI-specific 4 bp single-stranded overhangs. Therefore, the overall structure of each module in the PCR product (or synthesized polynucleotide) takes the form: 5'-BsmBI[CTCG]NNNN-TUM-NNNNtg[AGAC]BsmBI-3' ( Figure 11 B), which, after being cloned in BsmBI-mediated cloning, becomes 5'-BsaI[NNNN]-TUM-[NNNN]BsaI-3' in pML1 ( Figure 11 C).

[0284] Because each TUM is defined by the tetranucleotides flanking it, these module-specific bases effectively form the addressing system for each TUM, and they determine its position and orientation within the assembled TU. The developers of MoClo and GoldenBraid2.0 have collaborated to develop a common syntax or set of standard addresses (referred to as "fusion sites" in the MoClo system and "barcodes" in GoldenBraid2.0) for plant expression of a wide range of TUM species, to facilitate component interchangeability. 33And this standard is also adopted here for the MIDAS-based assembly of TU expressed in filamentous fungi. Figure 12 ).

[0285] Therefore, for filamentous fungal expression, the ProUTR module (containing a promoter, a 5' untranslated region (UTR), and an ATG start codon) has GGAG as a module-specific 5' nucleotide and ATG as a module-specific 3' nucleotide (i.e., 5'-GGAG-ProUTR-A). ATG -3'), where the translation start codon is underlined. Similarly, the CDS module has AATG and GCTT on both sides (i.e., 5'-A). ATG -CDS-GCTT-3'), while the UTRterm module (consisting of a 3' UTR and a 3' non-transcribed region, including a polyadenylation signal) has the form 5'-GCTT-UTRterm-CGCT-3'. The design considerations for PCR primers used to amplify these three types of TUM are shown in Table 12.

[0286] Following BsmBI-mediated assembly of TUM in pML1, the reaction was transformed into E. coli strains such as DH5α (or equivalents) and plated on LB agar plates supplemented with spectinomycin, IPTG, and X-Gal. Plasmids containing cloned TUM were identified by screening for white colonies and confirmed by sequencing.

[0287] At MIDAS Level 1, it is important to mask or remove all internal AarI, BsaI, and BsmBI recognition sites from TUM. The process of masking or removing these prohibited sites, known as “domestication,” can be achieved by: (i) excluding these sites when ordering the sequence from the gene synthesis formula, (ii) targeted mutagenesis, or (iii) using masking oligonucleotides that form a triplet with the target DNA, thereby preventing restriction enzyme cleavage. 30 Previously, IIS-type enzymes were used for mutations. 34 Purpose of domestication in Kinmen 27,35 Similarly, we tamed the MIDAS module by designing primers (referred to as taming primers) that overlapped with the internal IIS type restriction site and contained single nucleotide mismatches that disrupted said site. Since the PCR products were designed to assemble together in the MIDAS using a BsmBI-mediated Golden Gate reaction to form a full-length tamed TUM in pML1, it is important that the MIDAS taming primers were designed with BsmBI restriction sites that produce compatible single-stranded overhangs at the 5' end.

[0288] Level 2: TU Assembly

[0289] At level 2, compatible grouped clones and sequence-verified level 1 TUMs (e.g., ProUTR, CDS, and UTRterm modules) are assembled in the pML2 final vector using a BsaI-mediated Golden Gate reaction, resulting in a level 2 plasmid (pML2 initiation clone) containing a complete (i.e., full-length) eukaryotic TU. The module addressing criteria described earlier ensure that the assembly of TUs proceeds in an ordered, oriented manner, with the 3' end of one module compatible with the 5' end of the next module.

[0290] The module-specific bases GGAG at the 5' end of the ProUTR module and CGCT at the 3' end of the UTRterm module are compatible with the single-stranded overhangs generated by BsaI digestion of the pML2 final vector, and thus these bases define the outermost clonal boundary of the secondary assembly.

[0291] In MIDAS, there are eight level 2 (pML2) final vectors in which TUs can be assembled. The choice of vector depends on the desired configuration of the TUs in the multigene plasmid produced at level 3, namely: (i) the desired order in which the TUs are added to the multigene plasmid, (ii) the desired orientation of the multigene plasmid during assembly, and (iii) the desired orientation of each TU in the multigene plasmid. These characteristics are discussed further below.

[0292] The pML2 vectors differ from one another in the arrangement of specific nucleic acid sequence components that form the core of MIDAS operation. These sequence components are collectively referred to as the MIDAS box. Figure 13 The second-level assembly of TU was defined and the assembly of multigene constructs generated at the third level was controlled.

[0293] Each MIDAS box is defined by the following characteristics: (i) a Golden Gate clone box with divergent BsaI recognition sites on both sides, (ii) different arrangements of AarI and BsmBI recognition sites, and (iii) the presence or absence of the lacZα scoring marker. These characteristics are described in more detail.

[0294] Unlike typical Golden Gate cloning boxes (which usually contain the lacZα gene for blue / white selection), all eight pML2 vectors contain mutant *E. coli* pheS genes (driven by the promoter of the *E. coli* chloramphenicol acetyltransferase* gene) with divergent BsaI recognition sites flanking them. The *E. coli* pheS gene Thr... 251 Ala / Ala 294 The Gly double mutant provides high lethality to cells grown on LB medium supplemented with the phenylalanine analogue 4-chloro-phenylalanine 4CP. 36During BsaI-mediated secondary assembly of TU, ​​the mutant pheS gene is eliminated from the pML2 vector and can therefore be used as a negative selection marker.

[0295] The eight pML2 vectors, depending on the presence or absence of the lacZα gene in the MIDAS cassette, can be divided into two categories: "blue" and "white" (see [link to relevant documentation]). Figure 13 There are four “blue” pML2 vectors (indicated by “B” in the plasmid name) and four “white” pML2 vectors (indicated by “W” in the plasmid name). The “blue” and “white” vectors also differ in the relative configuration of the AarI and BsmBI restriction sites in their MIDAS boxes. Therefore, in the “blue” vector, the entire MIDAS box has converging BsmBI sites on both sides, and nested within it is the lacZα gene with divergent AarI sites on both sides. In the “white” vector, the enzyme configuration is reversed (the entire MIDAS box has converging AarI sites on both sides, and nested within it are two divergent BsmBI sites), and the lacZα gene is absent. It is important to note that the lacZα chromogenic marker in the pML2 vector is not used for blue / white selection during the second-level golden gate assembly of TU (it is reserved for third-level cloning), but the selection of the "blue" or "white" vector in which TU should be assembled must be made during the second-level assembly of TU, ​​as this will determine the order in which TU is added to the multigene construct at level 3. Similarly, the AarI and BsmBI sites are not used for the second-level assembly of TU; instead, they are integral components of the third-level assembly of the multigene construct. These considerations, including the differences between (+) and (-) vectors, are discussed further below in the description of level 3.

[0296] The orientation (transcriptional direction) of each TU can be freely determined by assembling each TU into a pML2 "forward" vector (indicated by "F" in the plasmid name) or a pML2 "reverse" vector (indicated by "R" in the plasmid name). The BsaI recognition site (for the golden gate assembly of the TU) in the pML2 "reverse" vector is swapped relative to the BsaI fusion site in the pML2 "forward" vector. Therefore, the pheS-based golden gate orientation of the pML2 "forward" vector is... The BsaI recognition sites of the pML2 "reverse" vector are swapped: The arrows indicate the transcription direction of the mutant pheS gene.

[0297] In contrast to the primary modules of the clone, when E. coli DH5α cells (or equivalents) transformed by the assembly reaction are plated on LB plates supplemented with kanamycin and 4CP, the pML2 final vector provides kanamycin resistance, allowing for efficient anti-selection against the primary module backbone, while the mutant pheS gene provides strong negative selection against any parental pML2 final plasmid.

[0298] Level 3: Assembly of multi-gene constructs

[0299] At MIDAS level 3, the TU sequence assembled in the pML2 plasmid is loaded (via binary assembly) into the level 3 final vector (pML3) to form a multigene construct.

[0300] At level 3, the assembly of the multi-gene construct hinges on the relative configuration of the AarI and BsmBI restriction sites in the MIDAS cassette located in the "blue" and "white" pML2 vectors; the nesting and inverted configuration of these restriction sites in the white vector compared to the blue vector is a defining characteristic of the MIDAS multi-gene assembly process. In the "blue" vector, the entire MIDAS cassette has converging BsmBI sites on both sides, and nests within it the lacZα gene with divergent AarI sites on both sides. In the "white" vector, the enzyme configuration is inverted (the entire MIDAS cassette has converging AarI sites on both sides and nests within it two divergent BsmBI sites), and the lacZα gene is absent. Figure 14 As shown, the nesting and inversion of restriction sites in the "blue" and "white" vectors means that TU assembled in the "white" MIDAS box can be inserted into the "blue" MIDAS box using an AarI-mediated gold gate reaction, and conversely, TU assembled in the "blue" MIDAS box can be cloned into the "white" MIDAS box using a BsmBI-mediated gold gate reaction. This cloning cycle (i.e., alternating between "white" and "blue" pML2 initiation clones) can be repeated indefinitely.

[0301] The Golden Gate cloning cassette present in the level 3 final vector pML3 consists of lacZα genes flanked by divergent AarI sites: [CATT]AarI-lacZα-AarI[CGTA]. Therefore, the MIDAS level 3 assembly is always initiated using an AarI-mediated Golden Gate reaction between pML3 and a TU already assembled in the pML2 “white” final vector (i.e., the first TU is always added). Figure 14The resulting plasmids, along with TU cloned into the pML2 "blue" final vector, were then used for BsmBI-mediated Golden Gate reactions. Additional TUs were added by alternating between AarI and BsmBI-mediated Golden Gate reactions, using pML2 "white" and pML2 "blue" initiating clones, respectively. Thus, each plasmid generated by cloning TU into the multigene construct became a final vector for the next round of TU addition. Figure 14 ).

[0302] After each cloning cycle, *E. coli* DH5α cells (or equivalents) were transformed using the Golden Gate reaction, plated on LB plates supplemented with spectinomycin, IPTG, and X-Ga1, and positive clones were identified by blue / white selection. Spectinomycin selected cells that had taken up the grade 3 plasmid and performed anti-selection against any pML2 plasmid backbone. Notably, although the lacZα chromogenic marker present in the pML2 “blue” vector was not previously used during grade 2 assembly of TUs, it is now used for blue / white selection at the multigene assembly level (grade 3). Therefore, for TUs assembled in the multigene constructs using the AarI-mediated Golden Gate reaction, white colonies were picked for analysis, while for TUs assembled in the multigene constructs using the BsmBI-mediated Golden Gate reaction, blue colonies were picked for analysis (see Table 13).

[0303] In its simplest configuration, MIDAS can achieve multi-genome assembly using only two pML2 final vectors: one "white" vector and one "blue" vector. Figure 15 A). However, the provision of the complete set of eight pML2 vectors enables maximum user control over the following characteristics: (i) the order in which each TU is added to the growing multigene construct, (ii) the desired orientation (i.e., transcription direction) of each TU, and (iii) the polarity of the assembly, i.e., the orientation in which the input TU is loaded into the multigene construct.

[0304] First, and as previously stated, the order in which each TU is added to the growing multigene construct is controlled by the selection of the “white” or “blue” pML2 final vector in which the TU is assembled.

[0305] Secondly, and as previously discussed in the discussion of secondary characteristics, the orientation (transcriptional direction) of the TU can be freely determined by the choice of a "forward" or "reverse" pML2 vector in which the TU is assembled. Expanding MIDAS to include the option of assembling the TU in either orientation, and extending the vector set to four pML2 plasmids (see [link to MIDAS]), further simplifies this process. Figure 13 and Figure 15 B).

[0306] Third, the polarity of the multigenome assembly (i.e., the direction in which the new TU is added to the growing multigenome assembly) can also be freely determined, in this case by assembling the TU in a pML2 final vector with "positive" (+) or "negative" (-) polarity. Figure 13 Using the pML2(+) introductory clone for level 3 assembly ensures that the next added TU will be in accordance with the Spec present in pML3. R Genes are added in the same direction as the transcription direction, meaning that the next assembled TU in the multigene construct will be added to the right of the TU added using the pML2(+) introductory clone (e.g., ...). Figure 15 A and Figure 15 (As shown in B). Instead, using the pML2(-) initiator clone for level 3 assembly forces the next TU to work with the Spec present in pML3. R Adding genes in opposite transcriptional directions results in the next TU loaded into the multigene construct being added to the left of the TU added using the pML2(-) introductory clone. However, if the multigene construct is built using introductory clones of both polarities (i.e., both pML2(+) and pML2(-)), this would provide MIDAS with the ability to switch the direction in which new TUs are added to the 3-level assembly, and for Figure 15 For the assumed assembly shown in C, all subsequently added TUs will be nested between TU3 and TU2.

[0307] bacterial and fungal strains

[0308] Routine growth of *E. coli* was carried out in LB broth at 37°C. Chemically competent *E. coli* HST08Stellar cells (Clontech Laboratories, Inc.) were used for routine plasmid transformation and maintenance. The *Penicillium paxilli* strain used in this study is shown in Table 7.

[0309] Solution for MIDAS Level 1 module cloning

[0310] The PCR amplification module was purified using a centrifugation column protocol and cloned into the MIDAS grade 1 plasmid pML1 via BsmBI-mediated Golden Gate assembly. Typically, 1–2 μL (approximately 50–200 ng) of micronized pML1 plasmid DNA was mixed with 1–2 μL of each purified PCR fragment, 1 μL of BsmBI (20 U / μL), 1 μL of T4 DNA ligase (20 U / μL), and 2 μL of 10×T4 DNA ligase buffer in a total reaction volume of 20 μL. The reaction was incubated at 37°C for 1–3 hours, and aliquots (typically 2–3 μL) were transformed into 30 μL of *E. coli* HST08 Stellar competent cells via heat shock. After the recovery period (i.e., adding 250 μL of SOC medium and incubating at 37°C for 1 hour), aliquots of the transformation mixture were plated onto LB agar plates supplemented with 50 μg / mL spectinomycin, 1 mM IPTG, and 50 μg / mL X-Ga1. The plates were incubated overnight at 37°C, and white colonies were selected for analysis.

[0311] Solution for MIDAS Level 2 TU silk packaging

[0312] Using modules cloned at stage 1, full-length TUs were assembled into MIDAS stage 2 plasmids via BsaI-mediated Golden Gate assembly. Typically, 40 fmol of pML2 plasmid DNA was mixed with 40 fmol of plasmid DNA from each stage 1 initiating clone, 1 μL of BsaI-HF (20 U / μL), 1 μL of T4 DNA ligase (20 U / μL), and 2 μL of 10×T4 DNA ligase buffer in a total reaction volume of 20 μL. The reaction was incubated in a DNA Engine PTC-200 Peltier thermal cycler (Bio-Rad) using the following parameters: 45 cycles (2 min at 37°C and 5 min at 16°C), followed by 5 min at 50°C and 10 min at 80°C. The reaction was transformed as described for stage 1 assembly and plated onto LB agar plates containing 75 μg / mL kanamycin and 1.25 mM 4CP. After incubation overnight at 37°C, colonies were picked for analysis.

[0313] Protocol for MIDAS Level 3 Multigen Assembly

[0314] The full-length TU assembled at stage 2 is used to generate multigenomic assemblies in the final stage 3 vector by alternating gold gates of AarI (for TU cloned in the pML2 "white" vector) or BsmBI (for TU cloned in the pML2 "blue" vector). Typically, 40 fmol of the final stage 3 vector plasmid DNA is mixed with 40 fmol of the stage 2 initiating clone plasmid DNA, 1 μL of BsaI-HF (20 U / μL), 1 μL of T4 DNA ligase (20 U / μL), and 2 μL of 10×T4 DNA ligase buffer, for a total reaction volume of 20 μL. The reaction is incubated in a DNA Engine PTC-200 Peltier thermal cycler (Bio-Rad) using the following parameters: 45 cycles (2 min at 37°C and 5 min at 16°C), followed by 5 min at 50°C and 10 min at 80°C. As described for Level 1 assembly, the reaction was transformed and plated onto LB agar plates supplemented with 50 μg / mL spectinomycin, 1 mM IPTG, and 50 μg / mL X-Gal. The plates were incubated overnight at 37°C. For AarI-mediated assembly reactions, white colonies were selected for analysis, while for BsmBI-mediated assembly reactions, blue colonies were selected.

[0315] Culture media and reagents used for fungal work

[0316] The CDYE (Czapex-Dox / yeast extract) medium containing trace elements was prepared using deionized water and contained 3.34% (w / v) Czapex-Dox (Oxoid Ltd., Hampshire, England), 0.5% (w / v) yeast extract (Oxoid Ltd., Hampshire, England), and 0.5% (v / v) trace element solution. For agar plates, Select agar (Invitrogen, California, USA) was added to bring the concentration to 1.5% (w / v).

[0317] The trace element solution was prepared in deionized water and contained 0.004% (w / v) cobalt(II) chloride hexahydrate (Ajax Finechem, Auckland, New Zealand), 0.005% (w / v) anhydrous copper(II) sulfate (Scharlau, Barcelona, ​​Spain), 0.05% (w / v) ferrous(II) sulfate heptahydrate (Merck, Darmstadt, Germany), 0.014% (w / v) manganese(II) sulfate tetrahydrate, and 0.05% (w / v) zinc sulfate heptahydrate (Merck, Darmstadt, Germany). The solution was preserved with 1 drop of 12M hydrochloric acid.

[0318] Regeneration (RG) medium was prepared using deionized water and contained 2% (w / v) malt extract (Oxoid Ltd., Hampshire, England), 2% (w / v) anhydrous D(+)-glucose (VWR International BVBA, Leuven, Belgium), 1% (w / v) fungal peptone (Oxoid Ltd., Hampshire, England), and 27.6% sucrose (ECPLtd. Birkenhead, Auckland, New Zealand). Select agar (Invitrogen, California, USA) was added to 1.5% or 0.8% (w / v) depending on whether the medium was for plate (1.5% RGA) or cover (0.8% RGA).

[0319] Fungal Protocol - Protoplast Preparation

[0320] Preparation of fungal protoplasts for transformation, according to Yelton et al. 37 And make modifications. Five aliquots of 25 mL of CDYE medium containing trace elements in 100 mL conical shake flasks were mixed with 5 × 10⁻⁶ molten metallurgical solution. 6Inoculate with spores and incubate at 28°C with shaking (200 rpm) for 28 hours. Filter the fermentation broth from all five shake flasks through a sterile diaper liner, and wash the combined mycelium three times with sterile water and once with OM buffer (10 mM Na₂HPO₄ and 1.2 M MgSO₄·7H₂O, adjusted to pH 5.8 with 100 mM NaH₂PO₄·2H₂O). Weigh the mycelium and resuspend each gram of mycelium in 10 mL of filtered, sterile lysin solution (prepared by resuspending 10 mg / mL of lysin (Sigma) from *Trichoderma harzianum* in OM buffer) and incubate at 30°C with shaking at 80 rpm for 16 hours. Filter the protoplasts through a sterile diaper liner into 250 mL Erlenmeyer flasks. Transfer 5 mL aliquots of filtered protoplasts to sterile 15 mL centrifuge tubes and cover with 2 mL ST buffer (0.6 M sorbitol and 0.1 M pH 8.0 Tris-HCl). Centrifuge the tubes at 2600 x g for 15 min at 4 °C. Transfer the white protoplast layer formed between the OM and ST buffer in each tube (2 mL aliquots) to sterile 15 mL centrifuge tubes, gently wash with a pipette in 5 mL STC buffer (1 M sorbitol, 50 mM pH 8.0 Tris-HCl and 50 mM CaCl2), and centrifuge at 2600 x g for 5 min at 4 °C. Decant the supernatant and combine the deposited protoplasts from multiple tubes in 5 mL aliquots in STC buffer by resuspending. Repeat the STC buffer wash three times until the protoplasts are combined in a single 15 mL centrifuge tube. The final protoplast deposit was resuspended in 500 μL of STC buffer, and the protoplast concentration was estimated using a hemocytometer. The protoplast stock solution was diluted to yield 1.25 × 10⁻⁶ per mL of STC buffer. 8 The final concentration of protoplasts was determined. Aliquots of protoplasts (100 μL) were immediately used for fungal transformation. Excess protoplasts were stored in 1.7 mL microcentrifuge tubes in 8% PEG solution (80 μL of protoplasts added to 20 μL of 40% (w / v) PEG 4000 in STC buffer) and stored at -80 °C.

[0321] Fungal Project - Transformation of Penicillium musculosus (P. paxilli)

[0322] From Vollmer and Yanosfsky 38 and Oliver, etc. 39Modified fungal transformation was performed in 100 μL (1.25 × 10⁻⁶) of freshly prepared STC buffer or stored in 8% PEG solution (as described above). 7 The transformation was performed in 1.7 mL microcentrifuge tubes containing protoplasts. A solution containing 2 μL spermidine (50 mM, in H₂O), 5 μL heparin (5 mg / mL, in STC buffer), and 5 μg plasmid DNA (250 μg / mL) was added to the protoplasts and incubated on ice for 30 min. Then, 900 μL of 40% PEG solution (40% (w / v) PEG 4000 in STC buffer) was added. The transformation mixture was incubated on ice for another 15–20 min, transferred to 17.5 mL of 0.8% RGA medium (preheated to 50°C) in a sterile 50 mL tube, mixed by inversion, and 3.5 mL aliquots were distributed onto 1.5% RGA plates. After incubation overnight at 25°C, 5 mL of 0.8% RGA (containing sufficient genistein to obtain a final concentration of 150 μg per mL of solid medium) was added to each plate. Incubate the plates at 25°C for 4 days. Pick spores from each colony and streak them onto CDYE agar plates supplemented with 150 μg / mL genimycin. Incubate the streaked plates at 25°C for 4 days. Suspend the spores from each colony in 50 μL of 0.01% (v / v) Triton X-100, and transfer 5 × 5 μL aliquots of the spore suspension to new CDYE agar plates supplemented with 150 μg / mL genimycin. Incubate the spore-forming plates at 25°C for 4 days and prepare the spore stock solution as described below. Suspend the colony plugs from the spore-forming plates in 2 mL of 0.01% (v / v) Triton X-100, and mix 800 μL of the suspended spores with 200 μL of 50% (w / v) glycerol in a 1.7 mL microcentrifuge tube. Inoculate 50 mL of CDYE medium with spore storage solution, then rapidly freeze in liquid nitrogen and store at -80°C.

[0323] Production and extraction of indole diterpenes

[0324] Fungal transformants were placed in 250 mL Erlenmeyer flasks lined with cotton lint and cultured in 50 mL of CDYE medium containing trace elements at 28 °C for 7 days with shaking (≥200 rpm). Mycelia were isolated from the fermentation broth by filtration through a diaper liner and transferred to 50 mL centrifuge tubes (Lab...). Thermo Fisher Scientific, and IDT was extracted by vigorous shaking (≥200 rpm) in 2-butanone for ≥45 minutes.

[0325] Thin layer chromatography

[0326] The supernatant of 2-butanone (containing the extracted indole diterpenes) was used for thin-layer chromatography (TLC) analysis on a solid-phase silica gel 60 aluminum plate (Merck). The indole diterpenes were chromatographically analyzed using chloroform:acetonitrile or dichloromethane:acetonitrile in a 9:1 ratio and visualized with Alder's reagent (1% (w / v) p-dimethylaminobenzaldehyde in 24% (v / v) HCl and 50% ethanol).

[0327] Liquid chromatography-mass spectrometry

[0328] Samples for liquid chromatography-mass spectrometry (LC-MS) were prepared from those transformants that tested positive by TLC. Therefore, 1 mL of 2-butanone supernatant (containing the extracted indole diterpenes) was transferred to a 1.7 mL microcentrifuge tube, and the 2-butanone was evaporated overnight. The contents were resuspended in 100% acetonitrile and filtered through a 0.2 μm membrane into an LC-MS vial. The LC-MS sample was then attached to... Chromatography was performed on a reverse-phase Thermo Scientific Accucore 2.6 μm C18 (50 × 2.1 mm) column in a Dionex 3000 standard LC system (Thermo Fisher Scientific) at a flow rate of 0.200 mL / min using a multi-step gradient method (Table 14), eluting with an aqueous solution of acetonitrile containing 0.01% formic acid. Mass spectra were obtained on maXis. TM Captured via online analysis on a Bruker quadrupole time-of-flight mass spectrometer.

[0329] Large-scale purification of indole diterpenes for NMR analysis

[0330] As described under "Production and Extraction of Indole Diterpenes," fungal transformants producing high levels of new indole diterpenes were grown in ≥1 L of CDYE medium containing trace elements. The mycelia were pooled in 1 L Schott flasks containing a stir bar. 2-Butanone was added and the indole diterpenes were extracted overnight with stirring (≥700 rpm). The extract was then passed through... 545(JT The crude extract was filtered, dried, and loaded onto silica gel with a rotary evaporator for crude purification via silica gel column chromatography, followed by final purification by semi-preparative HPLC. A 1 mL aliquot of the crude extract was injected into the silica gel column attached to the column chromatography plate. Semi-preparative reversed-phase Phenomenex 5μm C18 column (2) on a 3000 standard LC column (Dionex, Thermo Fisher Scientific) The solution was run in a 250 × 15 mm column at a flow rate of 8.00 mL / min. A multi-step gradient method was optimized for the purification of different groups of indole diterpenes. The purity of each indole diterpene was assessed by LC-MS, and the structure was identified by NMR.

[0331] NMR

[0332] NMR samples were prepared in deuterated chloroform. Compounds were analyzed by standard one-dimensional proton and carbon-13 NMR, two-dimensional correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC) spectroscopy, and heteronuclear multi-bond correlation (HMBC) spectroscopy.

[0333] Table 1-14, referenced in this specification, is described below:

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350] Industrial applicability

[0351] This invention has industrial applicability in the production of indole diterpenoids, particularly NA.

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[0391] (39) Oliver, R. P.; Roberts, I. N.; Harling, R.; Kenyon, L.; Punt, P. J.; Dingemanse, M. A.; van den Hondel, C. A. M. J. J. Curr. Genet. 1987, 12(3), 231 - 233. Sequence Listing <110> VAN DE BITTNER, Kyle Cornelius KESSANS, Sarah Adeline SCOTT, David Barry BUSTAMANTE RODRIGUEZ, Leyla Yolanda NICHOLSON, Matthew Joseph PARKER, Emily Jane VAN DOLLEWEERD, Craig John <120> Nodulisporic acid (nodulisporic acid) <130> 831107 JBA / rla <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 1825 <212> DNA <213> Hypoxylon policicidum <400> 1 tttgcccag atattccag cccggggttc gaaaacaaga gaaaacgctg gtatatgaat 60 tatatagcaa tcgtatgctg gcgcagaggc ttcatggctc atcggccgcc caggcagaag 120 tctcagattg aatcttgaca ttatagagga aattaccata cgcatctcca tcatcgcgaa 180 aggtttaccg aggcatccgt acacacccac agagaaaggg atgaatgcat ctgggttgag 240 agtcaagtgt gattggctcg tccatcgctc cgggatgaac tcatctggcc tgacgaaatt 300 tctcttatct aaagacggca gagttaaaat taagctcaaa ggatgactgt atcgaatcga 360 tactaacccc tgtgtatcgt gtaagtgggc atggatacga gcatatgctc tggtataaac 420 tggccatcaa ggatcactcc accaggaccg gtctcccgct gcgagccgct catcactgct 480 ggatataaac ggagtgcttc gttgatgcaa ctgttgaggt aaggcatatt gactagggtg 540 gaggttggca taggcagtcc acctatattc atgtcatcaa cctctttttg aagaattgcc 600 tgcttgtctg gatgggttgc cagaagaaac aagatggcag taagggtcga tgctttagag 660 tcgctaccgg ccacgacagc cagctcagaa tcatagacga ggtcatccgt atctttagac 720 tcgtcgtcta agagatggct gaatagatcg tgagtcatag ttggattctc cactgtgcgt 780 cagtcaggta tcctgattag tatgtaatta agtattacct acttgcttcc tttccactac 840 ctcctgtgag caccactgta gccaacttgt tgccttggac ctcaagaacg gtagattgcg 900 gagaaggatc aacgtccaag gcgcccacag aagtaacccc cctagttgct tgacagacct 960 cagttgcgtg aagccgggat gcgaaaggcc tttttgaatc atgttgaagg cctttccaaa 1020 tacaagccaa cccattgatt caaatgtgac tctttgcatc aattcagtta cttctaccgg 1080 ttgacctcct ctagatgaga gatagcccat gagttcttgg caacattgcc ggacatggcc 1140 ttcatgagct agcatagctg tatgctctat cagcatgaat cgcccacgcc agggttgtgt 1200 gatactcacc attaggactg aaggcctttt gccaaatttt cttccgtcgg aggtggaaat 1260 tcctgtccct tgtcatttga agggacttgt ggggataatt cacgtcgtag atggacccc 1320 tggcacactt ggccaaatcg cgaatggcat tgaagttgtt gacggatatc tcacgaggcc 1380 ctgacacata gtctatcac ttcgagttta tggcagagtt ggaataggc tagacttac 1440 taattcggac gaatcgcca tagacactat gcatattctg tactcagg tgccattttc 1500 tattacggc tgctgttttc atccatctca acgtgctcag cctggcggat accttgccgg 1560 ggaaatctct taagggc agaaaacc ggatataat gattgaaa cagatagaag 1620 agatagca tagccaaact tggatgaaca taaaagtgat cccatataat cccactgcca 1680 acaacgagca tatgagcgca atggggcaag tatgaagca atggacagg accggggcgt 1740 gcttgtccca ctctcctcgg atgataaaa tagtatgaga taggacggct gccaggcac 1800 tgatgcctaa atagctaa gtcat 1825 <210> 2 <211> 1599 <212> DNA <213> Hypoxylon policicidum <400> 2 ttatgcccag atatttccag cccggggttc gaaaacaaga gaaaacgctg gtatatgaat 60 tatatagcaa tcgtatgctg gcgcagaggc ttcatggctc atcggccgcc caggcagaag 120 tctcagattg aatcttgaca ttatagagga aattaccata cgcatctcca tcatcgcgaa 180 aggtttaccg aggcatccgt acacacccac agaaaggg atgaatgcat ctgggttgag 240 agtcaagtgt gattggctcg tccatcgctc cgggaatgaac tcatctggcc tgacgaaatt 300 tctcttatcc ctgtgtatcg tgtaagtggg catggatacg agcatatgct ctggtataaa 360 ctggccatca aggatcactc caccaggacc ggtctcccgc tgcgagccgc tcatcactgc 420 tggatataaa cggagtgctt cgttgatgca actgttgagg taaggcatat tgactagggt 480 ggaggttggc ataggcagtc cacctatatt catgtcatca acctcttttt gaagaattgc 540 ctgcttgtct ggatgggttg ccagaagaa caagatggca gtaagggtcg atgctgtaga 600 gtcgctaccg gccacgacag ccagctcaga atcatagacg aggtcatccg tatctttaga 660 ctcgtcgtct aagagatggc tgaatagatc gtgagtcata gttggatttt gcttcctttc 720 cactacctcc tgtgagcacc actgtagcca acttgttgcc ttggacctca agaacggtag 780 attgcggaga aggatcaacg tccaaggcgc ccacagaagt aaccccccta gttgcttgac 840 agacctcagt tgcgtgaagc cgggatgcga aaggcctttt tgaatcatgt tgaaggcctt 900 tccaaataca agccaaccca ttgattcaaa tgtgactctt tgcatcaatt cagttacttc 960 taccggttga cctctctag atgagagata gcccatgagt tcttggcaac attgccggac 1020 atggccttca tgagctagca tagcattagg actgaaggcc ttttgccaaa tttctttccg 1080 tcggaggtgg aaattcctgt cccttgtcat ttgaagggac ttgtggggat aattcacgtc 1140 gtagaatgga cccctggcac acttggccaa atcgcgaatg gcattgaagt tgttgacgga 1200 tatctcacga ggcctaattc ggacgaaatc gccatagaca ctatgcatat tctgtacttc 1260 aaggtgccat tttctatcta cggctgctgt tttcatccat ctcaacgtgc tcagcctggc 1320 ggataccttg ccggggaaat ctcttaaggg atgcagaaac aaccgatata taatgattga 1380 aagacagata gaagagatat agcatagcca aacttggatg aacataaaag tgatcccata 1440 taatcccact gccaacaacg agcatatgag cgcaatgggg caagtaatga agcaatggaa 1500 caggaccggg gcgtgcttgt cccactcc tcggatgaat aaatagtat gagataggac 1560 ggctgccagg caacgatgc ctaaaatagc windowcat 1599 <210> 3 <211> 532 <212> PRT <213> Hypoxylon policicidum <400> 3 Met Thr Leu Wing Ile Leu Gly Ile Ser Cys Leu Wing Wing Val Leu Ser 1 5 10 15 His Thr Ile Leu Phe Ile Arg Gly Glu Trp Asp Lys Ala Pro Val 20 25 30 Leu Phe His Cys Phe Ile Thr Cys Pro Ile Ala Leu Ile Cys Ser Leu 35 40 45 Leu Ala Val Gly Leu Tyr Gly Ile Thr Phe Met Phe Ile Gln Val Trp 50 55 60 Leu Cys Tyr Ile Ser Ser Ile Cys Leu Ser Ile Ile Tyr Arg Leu 65 70 75 80 Phe Leu His Pro Leu Arg Asp Phe Pro Gly Lys Val Ser Ala Arg Leu 85 90 95 Ser Thr Leu Arg Trp Met Lys Thr Ala Ala Val Asp Arg Lys Trp His 100 105 110 Leu Glu Val Gln Asn Met His Ser Val Tyr Gly Asp Phe Val Arg Ile 115 120 125 Arg Pro Arg Glu Ile Ser Val Asn Asn Phe Asn Ala Ile Arg Asp Leu 130 135 140 Ala Lys Cys Ala Arg Gly Pro Phe Tyr Asp Val Asn Tyr Pro His Lys 145 150 155 160 Ser Leu Gln Met Thr Arg Asp Arg Asn Phe His Leu Arg Arg Lys Lys 165 170 175 Ile Trp Gln Lys Ala Phe Ser Pro Asn Ala Met Leu Ala His Glu Gly 180 185 190 His Val Arg Gln Cys Cys Gln Glu Leu Met Gly Tyr Leu Ser Ser Arg 195 200 205 Gly Gly Gln Pro Val Glu Val Thr Glu Leu Met Gln Arg Val Thr Phe 210 215 220 Glu Ser Met Gly Trp Leu Val Phe Gly Lys Ala Phe Asn Met Ile Gln 225 230 235 240 Lys Gly Leu Ser His Pro Gly Phe Thr Gln Leu Arg Ser Val Lys Gln 245 250 255 Leu Gly Gly Leu Leu Leu Trp Ala Pro Trp Thr Leu Ile Leu Leu Arg 260 265 270 Asn Leu Pro Phe Leu Arg Ser Lys Ala Thr Ser Trp Leu Gln Trp Cys 275 280 285 Ser Gln Glu Val Val Glu Arg Lys Gln Asn Pro Thr Met Thr His Asp 290 295 300 Leu Phe Ser His Leu Leu Asp Asp Glu Ser Lys Asp Thr Asp Asp Leu 305 310 315 320 Val Tyr Asp Ser Glu Leu Ala Val Val Ala Gly Ser Asp Ser Thr Ala 325 330 335 Ser Thr Leu Thr Ala Ile Leu Phe Leu Leu Ala Thr His Pro Asp Lys 340 345 350 Gln Ala Ile Leu Gln Lys Glu Val Asp Asp Met Asn Ile Gly Gly Leu 355 360 365 Pro Met Pro Thr Ser Thr Leu Val Asn Met Pro Tyr Leu Asn Ser Cys 370 375 380 Ile Asn Glu Ala Leu Arg Leu Tyr Pro Ala Val Met Ser Gly Ser Gln 385 390 395 400 Arg Glu Thr Gly Pro Gly Gly Val Ile Leu Asp Gly Gln Phe Ile Pro 405 410 415 Glu His Met Leu Val Ser Met Pro Thr Tyr Thr Ile His Arg Asp Lys 420 425 430 Arg Asn Phe Val Arg Pro Asp Glu Phe Ile Pro Glu Arg Trp Thr Ser 435 440 445 Gln Ser His Leu Thr Leu Asn Pro Asp Ala Phe Ile Pro Phe Ser Val 450 455 460 Gly Val Tyr Gly Cys Leu Gly Lys Pro Phe Ala Met Met Glu Met Arg 465,470,475,480 Met Val Ile Ser Ser Ile Met Ser Arg Phe Asn Leu Arg Leu Leu Pro 485 490 495 Gly Arg Pro Met Ser His Glu Ala Ser Ala Pro Ala Tyr Asp Cys Tyr 500 505 510 Ile Ile His Ile Pro Ala Phe Ser Leu Val Phe Glu Pro Arg Ala Gly 515 520 525 Asn Ile Trp No 530 <210> 4 <211> 1826 <212> DNA <213> Hypoxylon policicidum <400> 4 atgtttgata ttgattttgg cattctattt ccaatttcct gggaacaatc ccctatcttt 60 ctggctgttg ggctaatatt cgcatttgcg accttatctc cgtggctccg ttccggggaa 120 cgcctgatca atggtcgcga aggcttcgaa atactgtgga cgaatgcgaa gaagcgatat 180 haaacgaagg ggcgatctgt tatggaggca ggctttca agttagata catcattgtc 240 tccaaatgaa agacatcaa tcgtgccggg tcatatgtgc tgatacttgg agatagtaca 300 acgattcttt ctacatgatg acggatagtg gtaccgaat ggtcctacac ccgagatacg 360 tcgacgagat ccggaacgac ccaaggttgg actttcacag atatatgaa actgtaatag 420 aattcgatcc attttctata tatccggggct gacgtctacg tacgcgaggt cctacatggg 480 ctcagacaga aaggtggcga gataaccga gatcttatcc aaaccaaatt gactagatct 540 gtgggtaagg ggttgtcgtc ctctagagaa tgcaggcaaa gggaaggaca agagtaact 600 aatgcccatc cttgtatagg taggctcatt gggtccatat cggctgaat cgaggacgcc 660 cttcacaatc gatgggaga aggcgaaggt gatatgaag agtaccttct agtgctccc 720 cctgctgaaa tgcatacaga atggcatgag attgtattgc tctccgtaat gatcccggtt 780 gttgcgcaag gagtatccaa atgtttgtc ggagatccat tgtgccgtaa caaggattgg 840 ataggtatga tcttaaggca tacaaggagt gtccagacag cactgcggtc ccttcgattg 900 tggccttact ttcttagacc acttgcagcc agatttctgc caacctgtcg acaagtcact 960 gcagaaatag aagaggccag gcgcatcatc aatccagtac ttgagaagaa gcgtgccgag 1020 aagctcgcaa tgattcagaa gggagaaaaa cctccagagc ccaacaccta catggattgg 1080 ttagaagaat ccgacaagga tgaattctat gacccagttg ttgctcagct gaagatttcc 1140 atggcagcta tacacgctac ctcggacctc ttatcgcaga caattttcag cctatgtgac 1200 agcccagagc tagtcaagga actgcgagcc gaagctgtgt ctgttatagg agcgtacggc 1260 tgggggaagg aagcaatcta caacctgaag ctgatggaca gcgtcctgaa ggagacgcaa 1320 cgcctaaaac ctatgcaaat cattaagcgc cggtgccgcc gtgtggactt gctatcgcta 1380 atcgctttgc ctttgcttgc tctctagact tgacgcgact agctctagat cgcataaaac 1440 tttccgatgg cacggttatc cctaggggct ccaaagttct tatctcctgc caacacatgt 1500 gggattcgaa cgtctacccc aacgcaaacc agtatgacgg tcatcgcttc tataaacttc 1560 ggcagcgcgc tggaatggag aattccgcac agctatcaac acctagtccg gatcatctgg 1620 gcttcggact gggaatgtat gcctgtcccg gtaggcatat cgcatcgacc gttatgaaag 1680 tcacactatg ccatatcctc ctgaagtatg acttcgaatt ggcggagggc tgcaccccta 1740 gagtgattga atatggtagt ttcttgttag ctgatccaac ggcgagagtt tctatcagac 1800 ggcggaaaga agagattcaa ctgtaa 1826 <210> 5 <211> 1536 <212> DNA <213> Hypoxylon policicidum <400> 5 atgtttgata ttgattttgg cattctattt ccaatttcct gggaacaatc ccctatcttt 60 ctggctgttg ggctaatatt cgcatttgcg accttatctc cgtggctccg ttccggggaa 120 cgcctgatca atggtcgcga aggcttcgaa atactgtgga cgaatgcgaa gaagcgatat 180 caaacgaagg ggcgatctgt tatggaggca ggctttcaa agtacaacga ttctttctac 240 atgatgacgg atagtggtac cgaaatggtc ctacacccga gatacgtcga cgagatccgg 300 aacgacccaa ggttggactt tcacagatat atgaaaacta aaggtggcga gataaaccga 360 gatcttatcc aaaccaaatt gactagatct gtgggtaggc tcattgggtc catatcggct 420 gaaatcgagg acgcccttca caatcgatgg gaagaaggcg aaggtgaata tgaagagtac 480 cttctagtgc ttccccctgc tgaaatgcat agaatggc atgagattgt attgctctcc 540 gtaatgatcc cggttgttgc gcaaggagat tccaaaatgt ttgtcggaga tccattgtgc 600 cgtaacaagg attggatagg tatgatctta aggcatacaa ggagtgtcca gacagcactg 660 cggtcccttc gattgtggcc ttactttctt agaccacttg cagccagatt tctgccaacc 720 tgtcgacaag tcactgcaga aataagaag gccaggcgca tcatcaatcc agtacttgag 780 aagaagcgtg ccgagaagct cgcaatgatt cagaagggag aaaaacctcc agagcccaac 840 acctacatgg attgggtaga agaatccgac areatgaat tctatgaccc agttgttgct 900 your feet tttccatggc agctatacac gctacctcgg acctctttatc gcagacaatt 960 ttcagcctat gtgacagccc agagctagtc agaactgc gagccgaagc tgtgtctgtt 1020 ataggagcgt acgggctgggg gaaggaagca atctacaacc tgaagctgat ggacagcgtc 1080 ctgaaggaga cgcaacgcct aaaacctatg caaatcaact tgacgcgact agctctagat 1140 cgcataaaac tttccgatgg cacggttatc cctaggggct ccaaagttct tatctcctgc 1200 cacaacatgt gggattcgaa cgtctacccc aacgcaaacc agtatgacgg tcatcgcttc 1260 tataaacttc ggcagcgc tggaatggag aattccgcac agctatcaac acctagtccg 1320 gatcatctgg gcttcggact gggaatgtat gcctgtcccg gtaggcatat cgcatcgacc 1380 gttatgaaag tcacactatg catatcctc ctgaagtatg acttcgaatt ggcggagggc 1440 tgcaccccta gagtgattga atatggtagt ttcttgttag ctgatccaac ggcgagagtt 1500 tctatcagac ggcggaaaga agagattcaa ctgtaa 1536 <210> 6 <211> 511 <212> PRT <213> Hypoxylon policicidum <400> 6 Met Phe Asp Ile Asp Phe Gly Ile Leu Phe Pro Ile Ser Trp Glu Gln 1 5 10 15 Ser Pro Ile Phe Leu Ala Val Gly Leu Ile Phe Ala Phe Ala Thr Leu 20 25 30 Ser Pro Trp Leu Arg Ser Gly Glu Arg Leu Ile Asn Gly Arg Glu Gly 35 40 45 Phe Glu Ile Leu Trp Thr Asn Ala Lys Lys Arg Tyr Gln Thr Lys Gly 50 55 60 Arg Ser Val Met Glu Ala Gly Phe Ser Lys Tyr Asn Asp Ser Phe Tyr 65 70 75 80 Met Met Thr Asp Ser Gly Thr Glu Met Val Leu His Pro Arg Tyr Val 85 90 95 Asp Glu Ile Arg Asn Asp Pro Arg Leu Asp Phe His Arg Tyr Met Lys 100 105 110 Thr Lys Gly Gly Glu Ile Asn Arg Asp Leu Ile Gln Thr Lys Leu Thr 115 120 125 Arg Ser Val Gly Arg Leu Ile Gly Ser Ile Ser Ala Glu Ile Glu Asp 130 135 140 Ala Leu His Asn Arg Trp Glu Glu Gly Glu Gly Glu Tyr Glu Glu Tyr 145 150 155 160 Leu Leu Val Leu Pro Pro Ala Glu Met His Thr Glu Trp His Glu Ile 165 170 175 Val Leu Leu Ser Val Met Ile Pro Val Val Ala Gln Gly Val Ser Lys 180 185 190 Met Phe Val Gly Asp Pro Leu Cys Arg Asn Lys Asp Trp Ile Gly Met 195 200 205 Ile Leu Arg His Thr Arg Ser Val Gln Thr Ala Leu Arg Ser Leu Arg 210 215 220 Leu Trp Pro Tyr Phe Leu Arg Pro Leu Ala Ala Arg Phe Leu Pro Thr 225 230 235 240 Cys Arg Gln Val Thr Ala Glu Ile Glu Glu Ala Arg Arg Ile Ile Asn 245 250 255 Pro Val Leu Glu Lys Lys Arg Ala Glu Lys Leu Ala Met Ile Gln Lys 260 265 270 Gly Glu Lys Pro Pro Glu Pro Asn Thr Tyr Met Asp Trp Leu Glu Glu 275 280 285 Ser Asp Lys Asp Glu Phe Tyr Asp Pro Val Val Ala Gln Leu Lys Ile 290 295 300 Ser Met Ala Ala Ile His Ala Thr Ser Asp Leu Leu Ser Gln Thr Ile 305 310 315 320 Phe Ser Leu Cys Asp Ser Pro Glu Leu Val Lys Glu Leu Arg Ala Glu 325 330 335 Ala Val Ser Val Ile Gly Ala Tyr Gly Trp Gly Lys Glu Ala Ile Tyr 340 345 350 Asn Leu Lys Leu Met Asp Ser Val Leu Lys Glu Thr Gln Arg Leu Lys 355 360 365 Pro Met Gln Ile Asn Leu Thr Arg Leu Ala Leu Asp Arg Ile Lys Leu 370 375 380 Ser Asp Gly Thr Val Ile Pro Arg Gly Ser Lys Val Leu Ile Ser Cys 385 390 395 400 His Asn Met Trp Asp Ser Asn Val Tyr Pro Asn Ala Asn Gln Tyr Asp 405 410 415 Gly His Arg Phe Tyr Lys Leu Arg Gln Arg Ala Gly Met Glu Asn Ser 420 425 430 Ala Gln Leu Ser Thr Pro Ser Pro Asp His Leu Gly Phe Gly Leu Gly 435 440 445 Met Tyr Ala Cys Pro Gly Arg His Ile Ala Ser Thr Val Met Lys Val 450 455 460 Thr Leu Cys His Ile Leu Leu Lys Tyr Asp Phe Glu Leu Ala Glu Gly 465 470 475 480 Cys Thr Pro Arg Val Ile Glu Tyr Gly Ser Phe Leu Leu Ala Asp Pro 485 490 495 Thr Ala Arg Val Ser Ile Arg Arg Arg Lys Glu Glu Ile Gln Leu 500 505 510 <210> 7 <211> 2012 <212> DNA <213> Hypoxylon pulicidicum <400> 7 ctaatctgcg atggaccctg accctaaccc tgaccctgac ctgaatccgg acccctgctc 60 atcctcgtct ctcacaatct cacggcgtag atctgccggc caagttggcg catcgactcc 120 gttccctctc gtgcccacat tatacttgag ttcaatcgcg tcgtccttga atcgagcgat 180 aggccttact tgtatgaacg tcgtgttcaa cgcgccggac cggaatgggc cgaaaaattc 240 cctttgaaag agaatctcgc cgtcgaattt gtacaatgcc gttagcagct taggcagcat 300 gtattgcatg gagtggtagt gtctgaaaac aggtgcgtcg tgacggcgga acaggtcagt 360 ccaggtatgc tcgtcgtatg tcccgactga tcgaacaagc cattgattgt aatagttgat 420 tgatacctgg aaatactgta tgagcgataa ccagcgatag cgtatccggg ggctacctac 480 atcaaccctg tagcttcccc ctttctcggc tctttggaca agggcgtcca agacagccgt 540 ggacccgcat attccggtac tagagatat gtcagtcagg ctgcaactct gctaccaagt 600 gaagaggtat ctctcgatcg gctcaccaat aatcagaatt gaacagagca ggagttacag 660 gctcttcaag acccatagca tggccgtacg aagtagaaat cccgcagcac gcatcgctaa 720 tctgctgcca gccgcttcta tggctccagg gtccatacca accatagcag ttctccctga 780 cgtaaataat accacgactg cggtcccgca ccagattgaa cacctcgtcg tagccaaagc 840 caaaacgttt catcacacct ggccgatagc tatctacaac cacatctgca tccaatatca 900 agtccctcag cttttggcgg tcagattcga catccaaccg tagccatgag ttccatttgc 960 cccaattcaa gtcttggtgt aacaccgaca tgtccgtgat gtctggggaa gtaacacgca 1020 ttacactagc ccccatctcc gcaagacttc gtgttatcgt cggacccgct atagcgcgcg 1080 ttaagtccac caccttcagc ccggccagcg gtctcttggg gctcgatgga gcgttaggat 1140 gatcaggcca ccaggctgct ggctgagaag aatttgggtc tctaatgacc tcatacagtc 1200 ccaccttgcc atttttctgg ccgtgctcgc tggcaaagta ctcagcggag ctgtagacaa 1260 tggtaccggc ttggcgatgt tcttcattta gcaaatggtc tagatgtgtg gcgtcatact 1320 ttgagagtct ttgttggatc ctgtcggtta cgacctcaga agtgtcttga acctcgcctt 1380 cgacgggcaa ccctagtgcc tgtagggtgg gctctggatt cattccccct ggaggaatat 1440 cgattagtct ctagattgac tcggcttgca agatcatacc gtgtgtatgg tagaaacggc 1500 catccttgt tttatagata ttggtaatca atgcccgttg tagagatgcg ttggcgcgat 1560 gtttatctcg gttcggaaaa agttctgctg tcagtgtatc aaatggcgag aaaggacgga 1620 cttctcctct atcctcgatt tgggtgagca tgggggacat gaggaacaag gttgcgtggt 1680 ctctatctga tgttagacaa acggccagtg cttcatatac atcacaatcc gagaacttac 1740 gtgtttattg agacattgct agggcgtaca ccatacttcc gatgggccag ataattcagg 1800 aacgttgctt caaaggcttt caaagctgag atgctctctg cgagcctcca gttaacagga 1860 atgctaggct tgacgtttcc ctcgaaggag acaaatttgg cgatttcggc cagctcaggg 1920 ggaagatttg gcattagcgg attctctaga atttcgcgta tgaatacaga ttccgcttgc 1980 ttgggaactg tattatccgc acttgactcc at 2012 <210> 8 <211> 1782 <212> DNA <213> Hypoxylon pulcicidum <400> 8 60. ctaatctgcg atggaccctg accctaacc tgaccctgac ctgaatccgg acccctgctc atcctcgtct ctcacaatct cacggcgtag atctgccggc caagttggcg catcgactcc gttccctctc gtgcccacat tatacttgag ttcaatcgcg tcgtccttga atcgagcgat 180 aggccttact tgtatgaacg tcgtgttcaa cgcgccggac cggaatgggc cgaaaaattc 240 cctttgaaag agaatctcgc cgtcgaattt gtacaatgcc gttagcagct taggcagcat gtattgcatg gagtggtagt gtctgaaaac aggtgcgtcg tgacggcgga acaggtcagt 360 ccaggtatgc tcgtcgtatg tcccgactga tcgaacaagc cattgattgt aatagttgat 420 tgatacatca accctgtagc ttcccccttt ctcggctctt tggacaaggg cgtccaagac 480 agccgtggac ccgcatattc cggtacaata atcagaattg aacagagcag gagttacagg ctcttcaaga cccatagcat ggccgtacga agtagaaatc ccgcagcacg catcgctaat ctgctgccag ccgcttctat ggctccaggg tccataccaa ccatagcagt tctccctgac gtaataata ccacgactgc ggtcccgcac cagattgac acctcgtcgt agccaaagcc aaaacgtttc atcacacctg gccgatagct atctacaacc acatctgcat ccaatatcaa gtccctcagc ttttggcggt cagattcgac atccaaccgt agccatgagt tccatttgcc 840 ccaattcaag tcttggtgta acaccgacat gtccgtgatg tctggggaag taacacgcat tacactagcc cccatctccg caagacttcg tgttatcgtc ggacccgcta tagcgcgcgt tagtccacc accttcagcc cggccagcgg tctcttgggg ctcgatggag cgttaggatg atcaggccac caggctgctg gctgagaga atttgggtct ctaatgacct catacagtcc caccttgcca tttttctggc cgtgctcgct ggcaaagtac tcagcggagc tgtagacaat ggtaccggct tggcgatgtt cttcatttag caaatggtct agatgtgtgg cgtcatactt tgagagtctt tgttggatcc tgtcggttac gacctcagaa gtgtcttgaa cctcgccttc gacgggcaac cctagtgcct gtagggtggg ctctggattc attcccccgt gtgtatggta 1320 gaaacggcca tcctttgttt tatagatatt ggtaatcaat gcccgttgta gagatgcgtt ggcgcgatgt ttatcggt tcggaaaaag ttctgctgtc agtgtatcaa atggcgagaa 1440 aggacggact tctcctctat cctcgatttg ggtgagcatg ggggacatga ggaacaaggt 1500 tgcgtggtct gtgtttattg agacattgct agggcgtaca catacttcc gatgggccag 1560 ataattcagg aacgttgctt caaaggcttt caaagctgag atgctctctg cgagcctcca 1620 gttaacagga atgctaggct tgacgtttcc ctcgaaggag acaaatttgg cgatttcggc 1680 cagctcaggg ggaagatttg gcattagcgg attctctaga atttcgcgta tgaatacaga 1740 ttccgcttgc ttgggaactg tattatccgc acttgactcc at 1782 <210> 9 <211> 593 <212> PRT <213> Hypoxylon policicidum <400> 9 Met Glu Ser Ser Ala Asp Asn Thr Val Pro Lys Gln Ala Glu Ser Val 1 5 10 15 Phe Ile Arg Glu Ile Leu Glu Asn Pro Leu Met Pro Asn Leu Pro Pro 20 25 30 Glu Leu Ala Glu Ile Ala Lys Phe Val Ser Phe Glu Gly Asn Val Lys 35 40 45 Pro Ser Ile Pro Val Asn Trp Arg Leu Ala Glu Ser Ile Ser Ala Leu 50 55 60 Lys Ala Phe Glu Ala Thr Phe Leu Asn Tyr Leu Ala His Arg Lys Tyr 65 70 75 80 Gly Val Arg Pro Ser Asn Val Ser Ile Asn Thr Asp His Ala Thr Leu 85 90 95 Phe Leu Met Ser Pro Met Leu Thr Gln Ile Glu Asp Arg Gly Glu Val 100 105 110 Arg Pro Phe Ser Pro Phe Asp Thr Leu Thr Ala Glu Leu Phe Pro Asn 115 120 125 Arg Asp Lys His Arg Ala Asn Ala Ser Leu Gln Arg Ala Leu Ile Thr 130 135 140 Asn Ile Tyr Lys Thr Lys Asp Gly Arg Phe Tyr His Thr His Gly Gly 145 150 155 160 Met Asn Pro Glu Pro Thr Leu Gln Ala Leu Gly Leu Pro Val Glu Gly 165 170 175 Glu Val Gln Asp Thr Ser Glu Val Val Thr Asp Arg Ile Gln Gln Arg 180 185 190 Leu Ser Lys Tyr Asp Ala Thr His Leu Asp His Leu Leu Asn Glu Glu 195 200 205 His Arg Gln Ala Gly Thr Ile Val Tyr Ser Ser Ala Glu Tyr Phe Ala 210 215 220 Ser Glu His Gly Gln Lys Asn Gly Lys Val Gly Leu Tyr Glu Val Ile 225 230 235 240 Arg Asp Pro Asn Ser Ser Gln Pro Ala Ala Trp Trp Pro Asp His Pro 245 250 255 Asn Ala Pro Ser Ser Pro Lys Arg Pro Leu Ala Gly Leu Lys Val Val 260 265 270 Asp Leu Thr Arg Ala Ile Ala Gly Pro Thr Ile Thr Arg Ser Leu Ala 275 280 285 Glu Met Gly Ala Ser Val Met Arg Val Thr Ser Pro Asp Ile Thr Asp 290 295 300 Met Ser Val Leu His Gln Asp Leu Asn Trp Gly Lys Trp Asn Ser Trp 305 310 315 320 Leu Arg Leu Asp Val Glu Ser Asp Arg Gln Lys Leu Arg Asp Leu Ile 325 330 335 Leu Asp Ala Asp Val Val Val Asp Ser Tyr Arg Pro Gly Val Met Lys 340 345 350 Arg Phe Gly Phe Gly Tyr Asp Glu Val Phe Asn Leu Val Arg Asp Arg 355 360 365 Ser Arg Gly Ile Ile Tyr Val Arg Glu Asn Cys Tyr Gly Trp Tyr Gly 370 375 380 Pro Trp Ser His Arg Ser Gly Trp Gln Gln Ile Ser Asp Ala Cys Cys 385 390 395 400 Gly Ile Ser Thr Ser Tyr Gly His Ala Met Gly Leu Glu Glu Pro Val 405 410 415 Thr Pro Ala Leu Phe Asn Ser Asp Tyr Cys Thr Gly Ile Cys Gly Ser 420 425 430 Thr Ala Val Leu Asp Ala Leu Val Gln Arg Ala Glu Lys Gly Gly Ser 435 440 445 Tyr Arg Val Asp Val Ser Ile Asn Tyr Tyr Asn Gln Trp Leu Val Arg 450 455 460 Ser Val Gly Thr Tyr Asp Glu His Thr Trp Thr Asp Leu Phe Arg Arg 465 470 475 480 His Asp Ala Pro Val Phe Arg His Tyr His Ser Met Gln Tyr Met Leu 485 490 495 Pro Lys Leu Leu Thr Ala Leu Tyr Lys Phe Asp Gly Glu Ile Leu Phe 500 505 510 Gln Arg Glu Phe Phe Gly Pro Phe Arg Ser Gly Ala Leu Asn Thr Thr 515 520 525 Phe Ile Gln Val Arg Pro Ile Ala Arg Phe Lys Asp Asp Ala Ile Glu 530 535 540 Leu Lys Tyr Asn Val Gly Thr Arg Gly Asn Gly Val Asp Ala Pro Thr 545,550,555,560 Trp Pro Ala Asp Leu Arg Arg Glu Ile Val Arg Asp Glu Asp Glu Gln 565,570,575 Gly Ser Gly Phe Arg Ser Gly Ser Gly Leu Gly Ser Gly Ser Ile Ala 580,585,590 Asp <210> 10 <211> 1577 <212> DNA <213> Hypoxylon policicidum <400> 10 atgtctaccc ctgagttcaa ggtgataatc gttggcggct cccttgcggg tctaacattg 60 gcccattgcc tcctccgcgc cggaatttcc cacatcgttc ttgagagacg tagtgtaatc 120 gcaccagagg aaggtgcctc aatagggatt cttcctaatg gcgctcgtgt cttggatcaa 180 ctaggcatct atgaacatat tcaagacact actgagccat tgagtacagc tcatatacga 240 tatccagatg ggttctattt cagcagtcgc tatcccgaga tcataaaaga aaggtgagca 300 ctcccatgaa catatgccta tcgtttgcgc aatggctgac cccagcatgg ccgttcgtag 360 gtttgggtat ccgatagcct ttttaccccg gaggagactt ttggagattc tctatacatc 420 gtaccctgac cattctaaca tctataccaa caagaatgtg atcaaggtgc aaagccatga 480 taatcaagtt tctgtcttga cagaggatgg aaatacatat cgaggggatc ttgtggtggg 540 cgccgacggt gtcaacagtc gagttctatc tgaaatttgg aaattagcag gtaatccctc 600 tctcacgaaa cgggaaggaa gaggtcagtc gagatcttat aatattcgca tagcgactca 660 tacagacgtt gataataaac ttaggaagga ccatcgagta cgcttgtgtg tttgggatat 720 cctcgccaat ctcagacctc aagcccggcg agcaggttaa cgcgttctac gacggactca 780 caatcgtcac gatccatggt agaaacggag agatattctg gttttttatc aagaaactct 840 cgcgcgcta tatctatccc gatctcatca gattacagca gaaagacgca gaagggatct 900 gtgaagaagc caagtcacta actgtctgga aaggtgttac attcggtgac atttgggaaa 960 gaagagaaac agcatcattg accgttttgg atgagttctt gcatcacact tggagttggg 1020 acagatcggt ttgtgtgggc gacagtatcc acaaggtata tgcctcctat ggtctgaagc 1080 acattaaggg gttggctaac gctgtcatat agatgactcc aaattttggc cagggtgcga 1140 atactgctat cgaggattct gccgctctag ccaatctact gcacagtttg atcaaggaga 1200 aacgagccga aaagccgact gactcagata tatcactgct tctgaggcag ttcaaatcac 1260 agcgtcttcg acgcgttcag aagatatata aaatgtcaag gtttgttacg cggcttcaag 1320 cgcgcgatgg gttgttgaat actcttctgg gacgccatta cgccccatat gcagcagatc 1380 ttccagctaa gattgcctct ggatgcatcg ctggtgcgga agttctggat tacctcccat 1440 tacccaaggt aactggggcg ggctgggaata ggggccatcg tagatctacc atgtacatcc 1500 tgctaggatt cacaggggta ttcacctctg ctctggcgat ggtagtgtta ctccatatta 1560 gagacatcgc ttcatag 1577 <210> 11 <211> 1392 <212> DNA <213> Hypoxylon pulicicidum <400> 11 atgtctaccc ctgagttcaa ggtgataatc gttggcggct cccttgcggg tctaacattg 60 gcccattgcc tcctccgcgc cggaatttcc cacatcgttc ttgagagacg tagtgtaatc 120 gcaccagagg aaggtgcctc aatagggatt cttcctaatg gcgctcgtgt cttggatcaa 180 ctaggcatct atgaacatat tcaagacact actgagccat tgagtacagc tcatatacga 240 tatccagatg ggttctattt cagcagtcgc tatcccgaga tcataaaga aaggtttggg 300 tatccgatag cctttttacc ccggaggaga cttttggaga ttctctatac atcgtaccct 360 gaccattcta acatctatac caacaagaat gtgatcaagg tgcaaagcca tgataatcaa 420 gtttctgtct tgacagagga tggaaataca tatcgagggg atcttgtggt gggcgccgac 480 ggtgtcaaca gtcgagttct atctgaaatt tggaaattag caggtaatcc ctctctcacg 540 aaacgggaag gaagagaag gaccatcgag tacgcttgtg tgtttgggat atcctcgcca 600 atctcagacc tcaagcccgg cgagcaggtt aacgcgttct acgacggact cacaatcgtc 660 acgatccatg gtagaaacgg agagatattc tggtttttta tcaagaaact ctcgcgtcgc 720 tatatctatc ccgatctcat cagattacag cagaaagacg cagaagggat ctgtgaagaa 780 gccaagtcac taactgtctg gaaaggtgtt acattcggtg acatttggga aagaagagaa 840 acagcatcat tgaccgtttt ggatgagttc ttgcatcaca cttggagttg ggacagatcg 900 gtttgtgtgg gcgacagtat ccacaagatg actccaaatt ttggccaggg tgcgaatact 960 gctatcgagg attctgccgc tctagccaat ctactgcaca gtttgatcaa ggagaaacga 1020 gccgaaaagc cgactgactc agatatatca ctgcttctga ggcagttcaa atcacagcgt 1080 cttcgacgcg ttcagaagat atataaaatg tcaaggtttg ttacgcggct tcaagcgcgc 1140 gatgggttgt tgaatactct tctgggacgc cattacgccc catatgcagc agatcttcca 1200 gctaagattg cctctggatg catcgctggt gcggaagttc tggattacct cccattaccc 1260 aaggtaactg gggcgggctg gaataggggc catcgtagat ctaccatgta catcctgcta 1320 ggattcacag gggtattcac ctctgctctg gcgatggtag tgttactcca tattagagac 1380 atcgcttcat ag 1392 <210> 12 <211> 463 <212> PRT <213> Hypoxylon policicidum <400> 12 Met Ser Thr Pro Glu Phe Lys Val Ile Ile Val Gly Gly Ser Leu Ala 1 5 10 15 Gly Leu Thr Leu Ala His Cys Leu Leu Arg Ala Gly Ile Ser His Ile 20 25 30 Val Leu Glu Arg Arg Ser Val Ile Ala Pro Glu Glu Gly Ala Ser Ile 35 40 45 Gly Ile Leu Pro Asn Gly Ala Arg Val Leu Asp Gln Leu Gly Ile Tyr 50 55 60 Glu His Ile Gln Asp Thr Thr Glu Pro Leu Ser Thr Ala His Ile Arg 65 70 75 80 Tyr Pro Asp Gly Phe Tyr Phe Ser Ser Arg Tyr Pro Glu Ile Ile Lys 85 90 95 Glu Arg Phe Gly Tyr Pro Ile Ala Phe Leu Pro Arg Arg Arg Leu Leu 100 105 110 Glu Ile Leu Tyr Thr Ser Tyr Pro Asp His Ser Asn Ile Tyr Thr Asn 115 120 125 Lys Asn Val Ile Lys Val Gln Ser His Asp Asn Gln Val Ser Val Leu 130 135 140 Thr Glu Asp Gly Asn Thr Tyr Arg Gly Asp Leu Val Val Gly Ala Asp 145 150 155 160 Gly Val Asn Ser Arg Val Leu Ser Glu Ile Trp Lys Leu Ala Gly Asn 165 170 175 Pro Ser Leu Thr Lys Arg Glu Gly Arg Gly Arg Thr Ile Glu Tyr Ala 180 185 190 Cys Val Phe Gly Ile Ser Ser Pro Ile Ser Asp Leu Lys Pro Gly Glu 195 200 205 Gln Val Asn Ala Phe Tyr Asp Gly Leu Thr Ile Val Thr Ile His Gly 210 215 220 Arg Asn Gly Glu Ile Phe Trp Phe Phe Ile Lys Lys Leu Ser Arg Arg 225 230 235 240 Tyr Ile Tyr Pro Asp Leu Ile Arg Leu Gln Gln Lys Asp Ala Glu Gly 245 250 255 Ile Cys Glu Glu Ala Lys Ser Leu Thr Val Trp Lys Gly Val Thr Phe 260 265 270 Gly Asp Ile Trp Glu Arg Arg Glu Thr Ala Ser Leu Thr Val Leu Asp 275 280 285 Glu Phe Leu His His Thr Trp Ser Trp Asp Arg Ser Val Cys Val Gly 290 295 300 Asp Ser Ile His Lys Met Thr Pro Asn Phe Gly Gln Gly Ala Asn Thr 305 310 315 320 Ala Ile Glu Asp Ser Ala Ala Leu Ala Asn Leu Leu His Ser Leu Ile 325 330 335 Lys Glu Lys Arg Ala Glu Lys Pro Thr Asp Ser Asp Ile Ser Leu Leu 340 345 350 Leu Arg Gln Phe Lys Ser Gln Arg Leu Arg Arg Val Gln Lys Ile Tyr 355 360 365 Lys Met Ser Arg Phe Val Thr Arg Leu Gln Ala Arg Asp Gly Leu Leu 370 375 380 Asn Thr Leu Leu Gly Arg His Tyr Ala Pro Tyr Ala Ala Asp Leu Pro 385 390 395 400 Ala Lys Ile Ala Ser Gly Cys Ile Ala Gly Ala Glu Val Leu Asp Tyr 405 410 415 Leu Pro Leu Pro Lys Val Thr Gly Ala Gly Trp Asn Arg Gly His Arg 420 425 430 Arg Ser Thr Met Tyr Ile Leu Leu Gly Phe Thr Gly Val Phe Thr Ser 435 440 445 Ala Leu Ala Met Val Val Leu Leu His Ile Arg Asp Ile Ala Ser 450 455 460 <210> 13 <211> 803 <212> Ms <213> Hypoxylon pulcicidum <400> 13 60. ttattgagcc ttccgcgcat tgcttcgttc ttggccatgt tcgtatttct tcacattcca cagacaaatt ccatagaggc tgtctaccgc aatgaataca ccgaggctcc atagtaccag aggactattg agccagttga atgcctccga ccagtacatg tacctcaata tggcaaaccc 240. aaccacacaa ccggatccta taaagcgaga taacctatac cttgttattt atatgtcctc aaagttctgt tgcataactt cggagcaaga agatggatta cctaccataa ggtatatgaa gctccacgac tactacttct ccctagaagc tggcagaacc ccccgacgct tagcagtagc 360 420. tggcaagcca ctgcacccca agaatatcca agtgctggcc caatctctgc tgctagggct agatggcccg tcaaggagcc caagacacct agtgcgaaaa tgagtgtgat gttgttcatc 480 acaagaggcg catgggccca ttcattcggc gcaaacctga tcgcggtgta catgactccg 540 agttgacta ccagtccagc tagaaacaca ccttgctcga tgcggctttt tgaagggtag ataagaccat ataccatttc ccaagcaaag ttgcagcaaa gagccagcgg tgccatgctg taagtctgtt cctggagcga gtgtagatc atgccggcgt agtttagg ccaacaaacg 720 cccattccga agacgaagat gtccgaaatc cattcgacgc gctgatattc aacgggtgca 780 ttggaacgat cgaatccatc cat 803 <210> 14 <211> 732 <212> DNA <213> Hypoxylon policicidum <400> 14 ttattgagcc ttccgcgcat tgctcgttc tggcatgt tcgtatttct tcacattcca 60 cagacaaatt ccatagaggc tgtctaccgc aatgaataca ccgaggctcc atagtaccag 120 aggactattg agccagttga atgcctccga ccagtacatg tacctcaata tggcaaaccc 180 aaccacacaa ccggatccta taaagcgaga taaccataag gtatatgaag ctccacgact 240 actactctc cctagaagct ggcagaaccc cccgacgctt agcagtagct ggcaagccac 300 tgcaccccaa gatatccaa gtgctggccc aatctctgct gctagggcta gatggcccgt 360 caaggagccc aagacaccta gtgcgaaaat gagtgtgatg ttgttcatca caaggcgc 420 atgggcccat tcattcggcg caaacctgat cgcggtgtac atgactccga gattgactac 480 cagtccagct agaacacac cttgctcgat gcggctttt gaagggtaga taagaccata 540 taccattcc caagcaagt tgcagcaag agccagcggt gccatgctgt aagtctgttc 600 ctggagcgag gtgtagatca tgccggcgta gtttatgagc caacaacgc ccattccgaa 660 gacgaagatg tccgaaatcc attcgacgcg ctgatattca acgggtgcat tggaacgatc 720 gatccatcc at 732 <210> 15 <211> 243 <212> PRT <213> Hypoxylon policicidum <400> 15 Met Asp Gly Phe Asp Arg Ser Asn Ala Pro Val Glu Tyr Gln Arg Val 1 5 10 15 Glu Trp Ile Ser Asp Ile Phe Val Phe Gly Met Gly Val Cys Trp Leu 20 25 30 Tyr Asn Only Gly With Tyr Thr Serves Leu Gln Glu Gln Thr Tyr 35 40 45 Ser Met Ala Pro Leu Ala Leu Cys Cys Asn Phe Ala Trp Glu Met Val 50 55 60 Tyr Gly Leu With Tyr Pro Ser Lys Ser Arg With Glu Gln Gly Val Phe 65 70 75 80 Leu Ala Gly Leu Val Val Asn Leu Gly Val Met Tyr Thr Ala Ile Arg 85 90 95 Phe Ala Pro Asn Glu Trp Ala His Ala Pro Leu Val Met Asn Asn Ile 100 105 110 Thr Leu Ile Phe Ala Leu Gly Val Leu Gly Ser Leu Thr Gly His Leu 115 120 125 Ala Leu Ala Ala Glu Ile Gly Pro Ala Leu Gly Tyr Ser Trp Gly Ala 130 135 140 Val Ala Cys Gln Leu Leu Leu Ser Val Gly Gly Phe Cys Gln Leu Leu 145 150 155 160 Gly Arg Ser Ser Ser Arg Gly Ala Ser Tyr Thr Leu Trp Leu Ser Arg 165 170 175 Phe Ile Gly Ser Gly Cys Val Val Gly Phe Ala Ile Leu Arg Tyr Met 180 185 190 Tyr Trp Ser Glu Ala Phe Asn Trp Leu Asn Ser Pro Leu Val Leu Trp 195 200 205 Ser Leu Gly Val Phe Ile Ala Val Asp Ser Leu Tyr Gly Ile Cys Leu 210 215 220 Trp Asn Val Lys Lys Tyr Glu His Gly Gln Glu Arg Ser Asn Ala Arg 225 230 235 240 Lys Ala Gln <210> 16 <211> 1514 <212> DNA <213> Hypoxylon policicidum <400> 16 atggaggttt cctcaagtga aactcccc attctatgga gaacgagtcc tccctcaaac 60 gactatgaaa atgcaagatg cagagtgttc aatggcaggc agcctgagca cttccccctt 120 gcaatagtca aggcaaacaa ggtcgagcat attgtagccg ccgtgaaact ggcagcagaa 180 ctagacgcct gtatcgccgt tcgttcgggc ggccacagcc tctcttgttg gactatacgc 240 catggggcga tccttattga cctcgaagat tatcagcact tgagctatga cgacgagatc 300 catgaagtgc aagcttcacc cagtacactt ggtgcggacc tacttacgtt ccttgcgaag 360 aaaaagaggt tctttcccgt aggtcactct ggagacattg gcttaggtgg ctatctcctt 420 cagggcggaa ttgggctcaa ttctcgggta tgaacgttga ccctacatac tcagtagctt 480 tcgaatactg acgatatctc tagggatatg ggtatgcctg tgaatacatt accgggcttg 540 atatcatcac cgctgatggt gaaatcaagc attgtgataa gacggaaaac tctgacctat 600 actgggctgc tcgtggagct ggaccgggta agttcctcaa agctgtctgt accagtgtgt 660 aagagttcta ataaagcaga attccctgca atcgttatac ggttcttcct gaagacgtgc 720 cccctcttgc cggtgtgtaa gcggagtaga tacgtctggc cggcggccat gtatgggaag 780 gtttttaagt ggcttgaaga ggtaagttta atcccaagga tctaggacgg caggggttaa tctgatccgc gtctttacag ctattgaatt ccttgagcga ggacgtcgag attgccgttt tcgggtttgt attgccccga ctcaaccagc caggcttagt cctccatgca acagcatttg 960 gtgactctag cgagaatgtc cgggaaaagc tcacgcctat catcaaaaat catcctccag ggactttctt ggctgaagat ttcgtgagca ccaacttccc cgaagactac gacttaggta 1080. aggataccat gccgcggggc gctcgctatt ttaccgacag cgtctttctc aagcccggta 1140 tcgacttcgt cgcaacctgc aaaggaatgt ttacggagct caaacatccc cgtgcattgg cgtactggca accgatgag accaatattg atcgcatcct tcccgatatg gcgatgagca tccatagtca tcattacgtg tcactacttg ccatctatga agaccccagt gaggaccaac agcaaatatc ctggatcata gatcgtatga aaagtcttga gccggcaatc ctgggaactt 1380 tcatagggga tgcacaccca gtggaaaggc catcaatta ttggtccgag gaagctgaag 1440 aacgggtgat cactattggg cggaagtggg accctagtag tagaattcgg ggtattgtgt 1500 tgagcgatgc ctaa 1514 <210> 17 <211> 1347 <212> DNA <213> Hypoxylon policicidum <400> 17 atggaggttt cctcaagtga aactcccc attctatgga gaacgagtcc tccctcaaac 60 gactatgaaa atgcaagatg cagagtgttc aatggcaggc agcctgagca cttccccctt 120 gcaatagtca aggcaaacaa ggtcgagcat attgtagccg ccgtgaaact ggcagcagaa 180 ctagacgcct gtatcgccgt tcgttcgggc ggccacagcc tctcttgttg gactatacgc 240 catggggcga tccttattga cctcgaagat tatcagcact tgagctatga cgacgagatc 300 catgaagtgc aagcttcacc cagtacactt ggtgcggacc tacttacgtt ccttgcgaag 360 aaaaagaggt tctttcccgt aggtcactct ggagacattg gcttaggtgg ctatctcctt 420 cagggcggaa ttgggctcaa ttctcgggga tatgggtatg cctgtgaata cattaccggg 480 cttgatatca tcaccgctga tggtgaaatc aagcattgtg ataagacgga aaactctgac 540 ctatactggg ctgctcgtgg agctggaccg gaattccctg caatcgttat acggttcttc 600 ctgaagacgt gccccctctt gccggtgtgt aagcggagta gatacgtctg gccggcggcc 660 atgtatggga aggtttttaa gtggcttgaa gagctattga attccttgag cgaggacgtc 720 gagattgccg ttttcgggtt tgtattgccc cgactcaacc agccaggctt agtcctccat 780 gcaacagcat ttggtgactc tagcgagaat gtccgggaaa agctcacgcc tatcatcaaa 840 aatcatcctc cagggacttt cttggctgaa gatttcgtga gcaccaactt ccccgaagac 900 tacgacttag gtaaggatac catgccgcgg ggcgctcgct attttaccga cagcgtcttt 960 ctcaagcccg gtatcgactt cgtcgcaacc tgcaaaggaa tgtttacgga gctcaaacat 1020 ccccgtgcat tggcgtactg gcaaccgatg aagaccaata ttgatcgcat ccttcccgat 1080 atggcgatga gcatccatag tcatcattac gtgtcactac ttgccatcta tgaagacccc 1140 agtgaggacc aacagcaat atcctggattc atagatcgta tgaaagtct tgagccggca 1200 atcctgggaa ctttcatagg ggatgcacac ccagtggaaa ggccatctaa ttattggtcc 1260 gaggaagctg aagaacggggt gatcactatt gggcggaagt gggaccctag tag1320 cggggtattg tgttgagcga tgcctaa 1347 <210> 18 <211> 448 <212> PRT <213> Hypoxylon policicidum <400> 18 Met Glu Val Ser Ser Glu Thr Leu Pro Ile Leu Trp Arg Thr Ser 1 5 10 15 Pro Pro Ser Asn Asp Tyr Glu Asn Ala Arg Cys Arg Val Phe Asn Gly 20 25 30 Arg Gln Pro Glu His Phe Pro Leu Ala Ile Val Lys Ala Asn Lys Val 35 40 45 Glu His Ile Val Val Val Val Lys Leu Val Hand Glu Leu Asp Val A Cys 50 55 60 Ile Ala Val Arg Serves Gly Gly His Serves Leu Serving Cys Trp Thr Ile Arg 65 70 75 80 His Gly Ala Ile Leu Ile Asp Leu Glu Asp Tyr Gln His Ser Tyr 85 90 95 Asp Asp Glu Ile His Glu Val Gln Ala Ser Pro Ser Thr Leu Gly Ala 100 105 110 Asp Leu Leu Thr Phe Leu Ala Lys Lys Lys Arg Phe Phe Pro Val Gly 115 120 125 His Ser Gly Asp Ile Gly Leu Gly Gly Tyr Leu Leu Gln Gly Gly Ile 130 135 140 Gly Leu Asn Ser Arg Gly Tyr Gly Tyr Ala Cys Glu Tyr Ile Thr Gly 145 150 155 160 Leu Asp Ile Ile Thr Ala Asp Gly Glu Ile Lys His Cys Asp Lys Thr 165 170 175 Glu Asn Ser Asp Leu Tyr Trp Ala Ala Arg Gly Ala Gly Pro Glu Phe 180 185 190 Pro Ala Ile Val Ile Arg Phe Phe Leu Lys Thr Cys Pro Leu Leu Pro 195 200 205 Val Cys Lys Arg Ser Arg Tyr Val Trp Pro Ala Ala Met Tyr Gly Lys 210 215 220 Val Phe Lys Trp Leu Glu Glu Leu Leu Asn Ser Leu Ser Glu Asp Val 225 230 235 240 Glu Ile Ala Val Phe Gly Phe Val Leu Pro Arg Leu Asn Gln Pro Gly 245 250 255 Leu Val Leu His Ala Thr Ala Phe Gly Asp Ser Ser Glu Asn Val Arg 260 265 270 Glu Lys Leu Thr Pro Ile Ile Lys Asn His Pro Pro Gly Thr Phe Leu 275 280 285 Ala Glu Asp Phe Val Ser Thr Asn Phe Pro Glu Asp Tyr Asp Leu Gly 290 295 300 Lys Asp Thr Met Pro Arg Gly Ala Arg Tyr Phe Thr Asp Ser Val Phe 305 310 315 320 Leu Lys Pro Gly Ile Asp Phe Val Ala Thr Cys Lys Gly Met Phe Thr 325 330 335 Glu Leu Lys His Pro Arg Ala Leu Ala Tyr Trp Gln Pro Met Lys Thr 340 345 350 Asn Ile Asp Arg Ile Leu Pro Asp Met Ala Met Ser Ile His Ser His 355 360 365 His Tyr Val Ser Leu Leu Ala Ile Tyr Glu Asp Pro Ser Glu Asp Gln 370 375 380 Gln Gln Ile Ser Trp Ile Ile Asp Arg Met Lys Ser Leu Glu Pro Ala 385 390 395 400 Ile Leu Gly Thr Phe Ile Gly Asp Ala His Pro Val Glu Arg Pro Ser 405 410 415 Asn Tyr Trp Ser Glu Glu Ala Glu Glu Arg Val Ile Thr Ile Gly Arg 420 425 430 Lys Trp Asp Pro Ser Ser Arg Ile Arg Gly Ile Val Leu Ser Asp Here 435 440 445 <210> 19 <211> 1694 <212> DNA <213> Hypoxylon policicidum <400> 19 ttacccaact agcagtggaa acttcgagtc gccttctgtg accctgaaat cccatttgtc 60 atccttgaat tcctttctct ctctgagagt caccatgaac tcccctgcag gtgtctgtat 120 ggaacttgca agtgttttca cgttggcctt gggaggaatc cactctccga gcacgggctc 180 catgtcaaac cgtaaaatca ccatggctac taccgctaat atctccccag atgcgaagtg 240 gcgcccagga cagatgttag gtgctgtccc gaaagactgg aacgcaacgc gtgtgacgtt 300 acggccactt cgcgatgtcc cactctccc ctggccgaga tatcgatatg cgtcgaagct 360 atcccctgtt tcgccccata ctgacttttc gcgattaaca catcttgcag ggatcacgat 420 cacggacccc ttcttgaata gaactcgctc gttgaggatg gtatcctcat ggaccatgcg 480 cgtgactata gcattagacc tcatacggag gacctcctga aaggtaccga gaagcaaact 540 gcacttggcg cgtacagcgc tgagatcaat agtacgaact atccctccat cggtacctgg 600 atgcgtgtac acagcagaag ccacgagctc ttgtcgaaga tccctcagca aactcgggcg 660 tgagtaaatt tcgaaaaggg tccaaaatgt ggaaggcgca gtattcgata ggacacctat 720 cgcgttaaca atttccattc gagctatatc ttgtgtggtg attcccttgt tatattggac 780 ctcccatcga ccatacgtca tttccgagga gttttcgtgg cctcccaggt tgtaatactc 840 tgttagagcg tccacaacct tacgacgatc atctagggac tttcgtgctg tgagaaaggg 900 aaggacattg aggattatca tattcatatt cgacgcgaaa tccctacacc acgtatgagc 960 tcagcaattc ttacacagca ttggggttct attctgtgct ttgacctacc aaaaagccct 1020 ttcaacttct atggatcgga atggattcat aggaccgtaa accgagtctg tgctcgccat 1080 tgtgatagaa cgtttacacc atgcaaataa gtcgaatgcc gcttgtttct ggctttggag 1140 ttcgtctatt gagggcttta tagcctcgat catggaaaga ttcatggcat ccaagccatg 1200 tcctataagt gcgtgatgca tagcatgttg tagctcatcc acgatactac ctttaagcaa 1260 tttaagtcca ggcccactga caccacctac acgttctgcg gccagtgtaa attcgaacgt 1320 atcgaatcgt atattctggg actgtctttg aatagccggg actagctcgc tagacgtgat 1380 cacataaagc cttgatagtg gcatctggag cgagaaggcg ggaagtccat atttgtcact 1440 atctacaccg tgtctatg tactatatatc agcatatctt cctcgcttca actaagcaac 1500 atgtagaagt cttaaccata cttgagcttg gaaaaatacc caaagccgtc agtcgcgata 1560 ccaagtatgt gcccgatgag tggtaccgta ggggatatca aacggggctc gcgggggtcg 1620 agagcagctc tccatcgagt accgtacagc aggatgcaaa aagcgagagt tatgataata 1680 acgataagtt ccat 1694 <210> 20 <211> 1545 <212> DNA <213> Hypoxylon policicidum <400> 20 ttacccaact agcagtggaa acttcgagtc gccttctgtg accctgaaat cccatttgtc 60 atccttgaat tcctttctct ctctgagagt caccatgaac tcccctgcag gtgtctgtat 120 ggaacttgca agtgttttca cgttggcctt gggaggaatc cactctccga gcacgggctc 180 catgtcaaac cgtaaaatca ccatggctac taccgctaat atctccccag atgcgaagtg 240 gcgcccagga cagatgttag gtgctgtccc gaaagactgg aacgcaacgc gtgtgacgtt 300 acggccactt cgcgatgtcc cactcttccc ctggccgaga tatcgatatg cgtcgaagct 360 atcccctgtt tcgccccata ctgacttttc gcgattaaca catcttgcag ggatcacgat 420 cacggacccc ttcttgaata gaactcgctc gttgaggatg gtatcctcat ggaccatgcg 480 cgtgactata gcattagacc tcatacggag gacctcctga aaggtaccga gaagcaaact 540 gcacttggcg cgtacagcgc tgagatcaat agtacgaact atccctccat cggtacctgg 600 atgcgtgtac acagcagaag ccacgagctc ttgtcgaaga tccctcagca aactcgggcg 660 tgagtaaatt tcgaaaaggg tccaaaatgt ggaaggcgca gtattcgata ggacacctat 720 cgcgttaaca atttccattc gagctatatc ttgtgtggtg attcccttgt tatattggac 780 ctcccatcga ccatacgtca tttccgagga gttttcgtgg cctcccaggt tgtaatactc 840 tgttagagcg tccacaacct tacgacgatc atctagggac tttcgtgctg tgagaaaggg 900 aaggacattg aggattatca tattcatatt cgacgcgaaa tcccaaaaag ccctttcaac 960 ttctatggat cggaatggat tcataggacc gtaaaccgag tctgtgctcg ccattgtgat 1020 agaacgttta caccatgcaa ataagtcgaa tgccgcttgt ttctggcttt ggagttcgtc 1080 tattgagggc tttatagcct cgatcatgga aagattcatg gcatccaagc catgtcctat 1140 aagtgcgtga tgcatagcat gttgtagctc atccacgata ctacctttaa gcaatttaag 1200 tccaggccca ctgacaccac ctacacgttc tgcggccagt gtaaattcga acgtatcgaa 1260 tcgtatattc tgggactgtc tttgaatagc cgggactagc tcgctagacg tgatcacata 1320 aagccttgat agtggcatct ggagcgagaa ggcgggaagt ccatatttgt cattgagctt 1380 ggaaaaatac ccaaagccgt cagtcgcgat accaagtatg tgcccgatga gtggtaccgt 1440 aggggatatc aaacggggct cgcgggggtc gagagcagct ctccatcgag taccgtacag 1500 caggatgcaa aaagcgagag ttatgataat aacgataagt tccat 1545 <210> 21 <211> 514 <212> PRT <213> Hypoxylon pulicicidum <400> 21 Met Glu Leu Ile Val Ile Ile Ile Thr Leu Ala Phe Cys Ile Leu Leu 1 5 10 15 Tyr Gly Thr Arg Trp Arg Ala Ala Leu Asp Pro Arg Glu Pro Arg Leu 20 25 30 Ile Ser Pro Thr Val Pro Leu Ile Gly His Ile Leu Gly Ile Ala Thr 35 40 45 Asp Gly Phe Gly Tyr Phe Ser Lys Leu Asn Asp Lys Tyr Gly Leu Pro 50 55 60 Ala Phe Ser Leu Gln Met Pro Leu Ser Arg Leu Tyr Val Ile Thr Ser 65 70 75 80 Ser Glu Leu Val Pro Ala Ile Gln Arg Gln Ser Gln Asn Ile Arg Phe 85 90 95 Asp Thr Phe Glu Phe Thr Leu Ala Ala Glu Arg Val Gly Gly Val Ser 100 105 110 Gly Pro Gly Leu Lys Leu Leu Lys Gly Ser Ile Val Asp Glu Leu Gln 115 120 125 His Ala Met His His Ala Leu Ile Gly His Gly Leu Asp Ala Met Asn 130 135 140 Leu Ser Met Ile Glu Ala Ile Lys Pro Ser Ile Asp Glu Leu Gln Ser 145 150 155 160 Gln Lys Gln Ala Ala Phe Asp Leu Phe Ala Trp Cys Lys Arg Ser Ile 165 170 175 Thr Met Ala Ser Thr Asp Ser Val Tyr Gly Pro Met Asn Pro Phe Arg 180 185 190 Ser Ile Glu Val Glu Arg Ala Phe Trp Asp Phe Ala Ser Asn Met Asn 195 200 205 Met Ile Ile Leu Asn Val Leu Pro Phe Leu Thr Ala Arg Lys Ser Leu 210 215 220 Asp Asp Arg Arg Lys Val Val Asp Ala Leu Thr Glu Tyr Tyr Asn Leu 225 230 235 240 Gly Gly His Glu Asn Ser Ser Glu Met Thr Tyr Gly Arg Trp Glu Val 245 250 255 Gln Tyr Asn Lys Gly Ile Thr Thr Gln Asp Ile Ala Arg Met Glu Ile 260 265 270 Val Asn Ala Ile Gly Val Leu Ser Asn Thr Ala Pro Ser Thr Phe Trp 275 280 285 Thr Leu Phe Glu Ile Tyr Ser Arg Pro Ser Leu Leu Arg Asp Leu Arg 290 295 300 Gln Glu Leu Val Ala Ser Ala Val Tyr Thr His Pro Gly Thr Asp Gly 305 310 315 320 Gly Ile Val Arg Thr Ile Asp Leu Ser Ala Val Arg Ala Lys Cys Ser 325 330 335 Leu Leu Leu Gly Thr Phe Gln Glu Val Leu Arg Met Arg Ser Asn Ala 340 345 350 Ile Val Thr Arg Met Val His Glu Asp Thr Ile Leu Asn Glu Arg Val 355 360 365 Leu Phe Lys Lys Gly Ser Val Ile Val Ile Pro Ala Arg Cys Val Asn 370 375 380 Arg Glu Lys Ser Val Trp Gly Glu Thr Gly Asp Ser Phe Asp Ala Tyr 385 390 395 400 Arg Tyr Leu Gly Gln Gly Lys Ser Gly Thr Ser Arg Ser Gly Arg Asn 405 410 415 Val Thr Arg Val Ala Phe Gln Ser Phe Gly Thr Ala Pro Asn Ile Cys 420 425 430 Pro Gly Arg His Phe Ala Ser Gly Glu Ile Leu Ala Val Val Ala Met 435 440 445 Val Ile Leu Arg Phe Asp Met Glu Pro Val Leu Gly Glu Trp Ile Pro 450 455 460 Pro Lys Ala Asn Val Lys Thr Leu Ala Ser Ser Ile Gln Thr Pro Ala 465 470 475 480 Gly Glu Phe Met Val Thr Leu Arg Glu Arg Lys Glu Phe Lys Asp Asp 485 490 495 Lys Trp Asp Phe Arg Val Thr Glu Gly Asp Ser Lys Phe Pro Leu Leu 500 505 510 Val Gly <210> 22 <211> 1125 <212> DNA <213> Hypoxylon pulicicidum <400> 22 atgtccttag gtttacagtg cttggcggca gtgttgtttt cggctttgtt ttcacttggg 60 gtcatcctag ttcatcttcc atggcgcgcc ttgaagtcaa aggacccgcg tgagcgaata 120 ttaggttcgc ccaaagaact ggttccaaca tgcccttacg aatatattcg aaatatatac 180 gggcgtcatc attgggcgcc ctttgtggcc aagttagcac cgaatctcaa agaaagtgat 240 tcagacaggt acacaatggt acttgaaatc atggactgca tacacctatg cctgattatg 300 gtcgatgatg tacgttcatc ttattatacg cctcttgttg gtgatagagg tgagagttgt 360 aaataactaa cccggactct cagattacag atgacagcga ctatcgtaaa ggccgcccag 420 cggcccatat catctatggc cgttcggaga cagctaaccg tgcttatctt cgtgtcagtc 480 agattataaa caagacaact caggacttcc cgcggctcgc cccgtgggtc acacagagtt 540 tggcagagat tctagagggc caggacatct cgctggtttg gcgacgagac ggcctcacta 600 gctttccaaa agctcacgac gagcgcgtga ttgcttatcg gtgcatgtca tctttgaaga 660 ctggcgcgct ttttaggttg ctagggaggc ttgtcttgga aaatcgttcc atggatgaca 720 cattgagtca ggttgggtaa gtgaactgta tccatcctga actcgacccc tttcacagtg 780 gcggaagcca atgctaaaac gtctagatac tattcacaat tacaaaatga ctgcaaaaat 840 gttttctcat ccgagtacgc aaaggcaaaa ggtactttag ctgaggactt acggaaccga 900 gagctgacgt atcctatcat cttggccctc aatgagcctg aaggatttta tattgagaaa 960 gcctttgagt ctggctcccc tcgtgacata caaaatgcaa tcggtgtaat acagagtgaa 1020 aacgtatacc gtgcttgttt ggacgagttg aaacaatatg aatcgaacgt cagagagtgg 1080 gttacactat ggggtagaaa ggagaaactc gatcttacgc attga 1125 <210> 23 <211> 981 <212> DNA <213> Hypoxylon policicidum <400> 23 atgtccttag gttacagtg cttggcggca gtgttgtttt cggctttgtt ttcacttggg 60 gtcatcctag ttcatcttcc atggcgcgcc ttgaagtcaa aggacccgcg tgagcgaata 120 ttaggttcgc ccaaagaact ggttccaaca tgcccttacg aatattcg aaatatatac 180 gggcgtcatc attgggcgcc ctttgtggcc aagttagcac cgaatctcaa agaaagtgat 240 tcagacaggt acacaatggt acttgaaatc atggactgca tacacctatg cctgattatg 300 gtcgatgata ttacagatga cagcgactat cgtaaaggcc gcccagcggc ccatatcatc 360 tatggccgtt cggagacagc taaccgtgct tatcttcgtg tcagtcagat tataaacaag 420 acaactcagg acttcccgg gctcgccccg tgggtcacac agagtttggc agagattcta 480 gagggccagg acatctcgct ggtttggcga cgagacggcc tcactagctt tccaaaagct 540 cacgacgagc gcgtgattgc ttatcggtgc atgtcatctt tgaagactgg cgcgcttttt 600 aggttgctag ggaggcttgt cttggaaaat cgttccatgg atgacacatt gagtcaggtt 660 ggatactatt cacaattaca aaatgactgc aaaaatgttt tctcatccga gtacgcaaag 720 gcaaaaggta ctttagctga ggacttacgg aaccgagagc tgacgtatcc tatcatcttg 780 gccctcaatg agcctgaagg attttatatt gagaaagcct ttgagtctgg ctcccctcgt 840 gacatacaaa atgcaatcgg tgtaatacag agtgaaaacg tataccgtgc ttgtttggac 900 gagttgaaac aatatgaatc gaacgtcaga gagtgggtta cactatgggg tagaaaggag 960 aaactcgatc ttacgcattg a 981 <210> 24 <211> 326 <212> PRT <213> Hypoxylon policicidum <400> 24 Put Ser Leu Gly Leu Gln Cys Leu Ala Ala Val Leu Phe Ser Ala Leu 1 5 10 15 Phe Ser Leu Gly Val Ile Leu Val His Leu Pro Trp Arg Ala Leu Lys 20 25 30 Ser Lys Asp Pro Arg Glu Arg Ile Leu Gly Ser Pro Lys Glu Leu Val 35 40 45 Pro Thr Cys Pro Tyr Glu Tyr Ile Arg Asn Ile Tyr Gly Arg His His 50 55 60 Trp Ala Pro Phe Val Ala Lys Leu Ala Pro Asn Leu Lys Glu Ser Asp 65 70 75 80 Ser Asp Arg Tyr Thr Met Val Leu Glu Ile Met Asp Cys Ile His Leu 85 90 95 Cys Leu Ile Met Val Asp Asp Ile Thr Asp Asp Ser Asp Tyr Arg Lys 100 105 110 Gly Arg Pro Ala Ala His Ile Ile Tyr Gly Arg Ser Glu Thr Ala Asn 115 120 125 Arg Ala Tyr Leu Arg Val Ser Gln Ile Ile Asn Lys Thr Thr Gln Asp 130 135 140 Phe Pro Arg Leu Ala Pro Trp Val Thr Gln Ser Leu Ala Glu Ile Leu 145 150 155 160 Glu Gly Gln Asp Ile Ser Leu Val Trp Arg Arg Asp Gly Leu Thr Ser 165 170 175 Phe Pro Lys Ala His Asp Glu Arg Val Ile Ala Tyr Arg Cys Met Ser 180 185 190 Ser Leu Lys Thr Gly Ala Leu Phe Arg Leu Leu Gly Arg Leu Val Leu 195 200 205 Glu Asn Arg Ser Met Asp Asp Thr Leu Ser Gln Val Gly Tyr Tyr Ser 210 215 220 Gln Leu Gln Asn Asp Cys Lys Asn Val Phe Ser Ser Glu Tyr Ala Lys 225 230 235 240 Ala Lys Gly Thr Leu Ala Glu Asp Leu Arg Asn Arg Glu Leu Thr Tyr 245 250 255 Pro Ile Ile Leu Ala Leu Asn Glu Pro Glu Gly Phe Tyr Ile Glu Lys 260 265 270 Ala Phe Glu Ser Gly Ser Pro Arg Asp Ile Gln Asn Ala Ile Gly Val 275 280 285 Ile Gln Ser Glu Asn Val Tyr Arg Ala Cys Leu Asp Glu Leu Lys Gln 290 295 300 Tyr Glu Ser Asn Val Arg Glu Trp Val Thr Leu Trp Gly Arg Lys Glu 305 310 315 320 Lys Leu Asp Leu Thr His 325 <210> 25 <211> 1439 <212> DNA <213> Hypoxylon pulicicidum <400> 25 ttaagagatg ttggtggcga cgtactcctt tccaccacga attgctttga cgaccttgtc 60 atattcctcg taggcatgct cctggctgtt gtgctctgaa acccagtccc taagtggcac 120 ttctgggtac atgttgatgc cctggtccgg ctgttccacg ttgtgctgaa gagctgaagc 180 tcgtggcttt cttttcggga gggaaagaaa tatgagttag ctaccgtacg tatctacgat 240 gttcttattg ttgttagact gctttacgta cctgattttc tcagccacac ggagcgcgag 300 agggacgttc ccctttcctg cgagctcgag gcagattgcc acgactgcgg catcctcaat 360 gacaagagac tccccaatgg gagagtttag ggcggaaggg tgcgaagcgt cgccaatgag 420 aatcatgcgg ccctgaggag agacccatga tggaagctgt cccagctgaa gaacgggttc 480 atcaacaagg cttccagatg gcgcggcctg gacgaccgct tcgatgttgg ctttgaacgg 540 ccatgattgg atgggcttga ggaagtcttg tgcggtagca ggagttgatg acgtcttggc 600 tgagttagga gattcctaga agatactata agcaggtgct cgtccaacat gtacctcaag 660 aatacatacc accttgtaca tgttactaaa tgctacgttg cgaccacccc cacaagtgac 720 gatggtgaga caggcagttg gaccaggcac aaatatcatc tggtctactt cacccgctcc 780 ttggaggatc cattgggcgt ctgggtttcc ttttaggcta gcgatatcag ccctaccccg 840 gatgtgtgcg tatccgctgt gggttggctt gaggctggag cctgcgacgg agtctcggcc 900 tttgctgttc acaccatctg cccagatgat gcagtctgcc tgcagcttct ggccattggc 960 agagacgccg gcctgatcgc tctcctccca gtaatcagag atctcagttc cgaaccgcat 1020 gtcgattcca agagtcttcg catgatcgta aacgatcttg gtcagctccc gtcggaggat 1080 caggtacttg ttgctcgatg gtttccgaac ttcaatggtc tgaacctgct ttccggtggt 1140 gtcatacaca gatgcttggg tgttattaaa cttccaagca ttcaaggcct gacccacagc 1200 tccgttgccc catttagaga caatcacacc cgcattgttg tcaatcatga cggtgtcatc 1260 tattatccta gtcattaaat tgctcttgat agtgtgagtg tatacttacc gacgtaattg 1320 agatccttgg accgttccag tgcaatgaca gaatgcccct tttggtgaca ttctatcgct 1380 gtggtaagac cagccaagcc aagtccaacc acaattacac taacaccggt tgaagccat 1439 <210> 26 <211> 1296 <212> DNA <213> Hypoxylon pulicicidum <400> 26 ttaagagatg ttggtggcga cgtactcctt tccaccacga attgctttga cgaccttgtc 60 atattcctcg taggcatgct cctggctgtt gtgctctgaa acccagtccc taagtggcac 120 ttctgggtac atgttgatgc cctggtccgg ctgttccacg ttgtgctgaa gagctgaagc 180 tcgtgggatg ttcttattgt tgttagactg ctttacgtac ctgattttct cagccacacg 240 gagcgcgaga gggacgttcc cctttcctgc gagctcgagg cagattgcca cgactgcggc 300 atcctcaatg acaagagact ccccaatggg agagtttagg gcggaagggt gcgaagcgtc 360 gccaatgaga atcatgcggc cctgaggaga gacccatgat ggaagctgtc ccagctgaag 420 aacgggttca tcaacaaggc ttccagatgg cgcggcctgg acgaccgctt cgatgttggc 480 tttgaacggc catgattgga tgggcttgag gaagtcttgt gcggtagcag gagttgatga 540 cgtcttggct gagttaggag attccacctt gtacatgtta ctaaatgcta cgttgcgacc 600 acccccacaa gtgacgatgg tgagacaggc agttggacca ggcacaaata tcatctggtc 660 tacttcaccc gctccttgga ggatccattg ggcgtctggg tttcctttta ggctagcgat 720 atcagcccta ccccggatgt gtgcgtatcc gctgtgggtt ggcttgaggc tggagcctgc 780 gacggagtct cggcctttgc tgttcacacc atctgcccag atgatgcagt ctgcctgcag 840 cttctggcca ttggcagaga cgccggcctg atcgctctcc tcccagtaat cagagatctc 900 agttccgaac cgcatgtcga ttccaagagt cttcgcatga tcgtaaacga tcttggtcag 960 ctcccgtcgg aggatcaggt acttgttgct cgatggtttc cgaacttcaa tggtctgaac 1020 ctgctttccg gtggtgtcat acacagatgc ttgggtgtta ttaaacttcc aagcattcaa 1080 ggcctgaccc acagctccgt tgccccattt agagacaatc acacccgcat tgttgtcaat 1140 catgacggtg tcatctatta tcttaccgac gtaattgaga tccttggacc gttccagtgc 1200 aatgacagaa tgcccctttt ggtgacattc tatcgctgtg gtaagaccag ccaagccaag 1260 tccaaccaca attacactaa caccggttga agccat 1296 <210> 27 <211> 431 <212> PRT <213> Hypoxylon pulicicidum <400> 27 Met Ala Ser Thr Gly Val Ser Val Ile Val Val Gly Leu Gly Leu Ala 1 5 10 15 Gly Leu Thr Thr Ala Ile Glu Cys His Gln Lys Gly His Ser Val Ile 20 25 30 Ala Leu Glu Arg Ser Lys Asp Leu Asn Tyr Val Gly Lys Ile Ile Asp 35 40 45 Asp Thr Val Met Ile Asp Asn Asn Ala Gly Val Ile Val Ser Lys Trp 50 55 60 Gly Asn Gly Ala Val Gly Gln Ala Leu Asn Ala Trp Lys Phe Asn Asn 65 70 75 80 Thr Gln Ala Ser Val Tyr Asp Thr Thr Gly Lys Gln Val Gln Thr Ile 85 90 95 Glu Val Arg Lys Pro Ser Ser Asn Lys Tyr Leu Ile Leu Arg Arg Glu 100 105 110 Leu Thr Lys Ile Val Tyr Asp His Ala Lys Thr Leu Gly Ile Asp Met 115 120 125 Arg Phe Gly Thr Glu Ile Ser Asp Tyr Trp Glu Glu Ser Asp Gln Ala 130 135 140 Gly Val Ser Ala Asn Gly Gln Lys Leu Gln Ala Asp Cys Ile Ile Trp 145 150 155 160 Ala Asp Gly Val Asn Ser Lys Gly Arg Asp Ser Val Ala Gly Ser Ser 165 170 175 Leu Lys Pro Thr His Ser Gly Tyr Ala His Ile Arg Gly Arg Ala Asp 180 185 190 Ile Ala Ser Leu Lys Gly Asn Pro Asp Ala Gln Trp Ile Leu Gln Gly 195 200 205 Ala Gly Glu Val Asp Gln Met Ile Phe Val Pro Gly Pro Thr Ala Cys 210 215 220 Leu Thr Ile Val Thr Cys Gly Gly Gly Arg Asn Val Ala Phe Ser Asn 225 230 235 240 Met Tyr Lys Val Glu Ser Pro Asn Ser Ala Lys Thr Ser Ser Thr Pro 245 250 255 Ala Thr Ala Gln Asp Phe Leu Lys Pro Ile Gln Ser Trp Pro Phe Lys 260 265 270 Ala Asn Ile Glu Ala Val Val Gln Ala Ala Pro Ser Gly Ser Leu Val 275 280 285 Asp Glu Pro Val Leu Gln Leu Gly Gln Leu Pro Ser Trp Val Ser Pro 290 295 300 Gln Gly Arg Met Ile Leu Ile Gly Asp Ala Ser His Pro Ser Ala Leu 305 310 315 320 Asn Ser Pro Ile Gly Glu Ser Leu Val Ile Glu Asp Ala Ala Val Val 325 330 335 Ala Ile Cys Leu Glu Leu Ala Gly Lys Gly Asn Val Pro Leu Ala Leu 340 345 350 Arg Val Ala Glu Lys Ile Arg Tyr Val Lys Gln Ser Asn Asn Asn Lys 355 360 365 Asn Ile Pro Arg Ala Ser Ala Leu Gln His Asn Val Glu Gln Pro Asp 370 375 380 Gln Gly Ile Asn Met Tyr Pro Glu Val Pro Leu Arg Asp Trp Val Ser 385 390 395 400 Glu His Asn Ser Gln Glu His Ala Tyr Glu Glu Tyr Asp Lys Val Val 405 410 415 Lys Ala Ile Arg Gly Gly Lys Glu Tyr Val Ala Thr Asn Ile Ser 420 425 430 <210> 28 <211> 1372 <212> DNA <213> Hypoxylon pulicicidum <400> 28 atggactcta ccgggagaca gcctctcagc caggacgggc agccatggca agctcttgcc 60 tctggcttgg gatttccaga tgaagaccag aagtattggt ggtcagtcat ggcgccccta 120 ttaggggaac tgatgaaatg ggccaactat cctgttgata aacagtatct tgttcttgcg 180 ttctgccatg aatacatact tccattttgc ggacctcgtc caacggccga aggcggtatc 240 ttctggccca cgttgatcac caaggatggt actccgttcg agccgagcct taacttctac 300 aaaaaaaag ccactctacg agttggatat gcgcctgcat gcgagctttc aggaagcaat 360 gacgacccta tcaatcaacg agccccaatt gcggcattgg agcaccaaaa gagagtccta 420 ccacagcaga atctcaaatg ggtggataat ttcaagaaag catggttcat cgataatgac 480 gacgcagtag ccttgaaggc acgcgtacat aacgagctct ttgaacaggc agccgtccaa 540 tgcttgatag gttatgtgtt ttccgactac acgcaggtca aggttgccat gagtcctctt 600 tggaagtcag tccagacagg ccagcaaatt tcgcgagtga tctgggacac ctttcgccag 660 cttggagacg atgcttccag ttacctagat tgcctatcag tgctagag gtatactgag 720 tctaaacagg ccaaactagc acaagtacag ccttccttcg tcaatttcga tgtgaatctg 780 aagggggact accagcagtc gcgacttaag gtatactatg ctacaccatg cactgcattc 840 gacacgatgg tccaggtttt cactttgggc ggaaggctca agggccctga agttgaccat 900 gcgattgaat gccttcgtgt cctatggccc agcgtgcttg cagttcctga aaaccatccg 960 gacgaccaag acttgcctcg acggtaccac tctgtagcag tgacccagtt taacttcgag 1020 ctctggccgg gagcgaaact gcctgttccc cagatctatc tcccgactaa cttttacggt 1080 cgtgatgaat tggaaattgc ggagggccta gagggatttt tcaagactct tggctggagt 1140 gagcctttcc atgcctataa acagaattac attgcaacat ggttagcta tcgcctttct 1200 ctcgctcatt gccctgatcc aatgaagtat tttactcaca cgatgtagtg ccacgcccga 1260 agggaaatgg aaagcgatcc aacacgacgt atcattctcg ttcaaggact caagcccata 1320 tgtgtcggtt tattataaac cagagctctc ggtcctatcc tcaccgtcgt ag 1372 <210> 29 <211> 1305 <212> DNA <213> Hypoxylon policicidum <400> 29 atggactcta ccgggagaca gcctctcagc caggacgggc agccatggca agctcttgcc 60 tctggcttgg gatttccaga tgaagaccag aagtattggt ggtcagtcat ggcgccccta 120 ttaggggaac tgatgaaatg ggccaactat cctgttgata aacagtatct tgttcttgcg 180 ttctgccatg aatacatact tccattttgc ggacctcgtc caacggccga aggcggtatc 240 ttctggccca cgttgatcac caaggatggt actccgttcg agccgagcct taacttctac 300 aaaaaaaag ccactctacg agttggatat gcgcctgcat gcgagctttc aggaagcaat 360 gacgacccta tcaatcaacg agccccaatt gcggcattgg agcaccaaaa gagagtccta 420 ccacagcaga atctcaaatg ggtggataat ttcaagaaag catggttcat cgataatgac 480 gacgcagtag ccttgaaggc acgcgtacat aacgagctct ttgaacaggc agccgtccaa 540 tgcttgatag gttatgtgtt ttccgactac acgcaggtca aggttgccat gagtcctctt 600 tggaagtcag tccagacagg ccagcaaatt tcgcgagtga tctgggacac ctttcgccag 660 cttggagacg atgcttccag ttacctagat tgcctatcag tgctagagga gtatactgag 720 tctaaacagg ccaaactagc acaagtacag ccttccttcg tcaatttcga tgtgaatctg 780 aagggggact accagcagtc gcgacttaag gtatactatg ctacaccatg cactgcattc 840 gacacgatgg tccaggtttt cactttgggc ggaaggctca agggccctga agttgaccat 900 gcgattgaat gccttcgtgt cctatggccc agcgtgcttg cagttcctga aaaccatccg 960 gacgaccaag acttgcctcg acggtaccac tctgtagcag tgacccagtt taacttcgag 1020 ctctggccgg gagcgaaact gcctgttccc cagatctatc tcccgactaa cttttacggt 1080 cgtgatgaat tggaaattgc ggagggccta gagggatttt tcaagactct tggctggagt 1140 gagcctttcc atgcctataa acagaattac attgcaacat gtgccacgcc cgaagggaaa 1200 tggaaagcga tccaacacga cgtatcattc tcgttcaagg actcaagccc atatgtgtcg 1260 gtttatta aaccagagct ctcggtccta tcctcaccgt cgtag 1305 <210> 30 <211> 434 <212> PRT <213> Hypoxylon policicidum <400> 30 Met Asp Ser Thr Gly Arg Gln Pro Leu Ser Gln Asp Gly Gln Pro Trp 1 5 10 15 Gln Ala Leu Ala Ser Gly Leu Gly Phe Pro Asp Glu Asp Gln Lys Tyr 20 25 30 Trp Trp Ser Val Met Ala Pro Leu Leu Gly Glu Leu Met Lys Trp Ala 35 40 45 Asn Tyr Pro Val Asp Lys Gln Tyr Leu Val Leu Ala Phe Cys His Glu 50 55 60 Tyr Ile Leu Pro Phe Cys Gly Pro Arg Pro Thr Ala Glu Gly Gly Ile 65 70 75 80 Phe Trp Pro Thr Leu Ile Thr Lys Asp Gly Thr Pro Phe Glu Pro Ser 85 90 95 Leu Asn Phe Tyr Lys Asn Lys Ala Thr Leu Arg Val Gly Tyr Ala Pro 100 105 110 Ala Cys Glu Leu Ser Gly Ser Asn Asp Asp Pro Ile Asn Gln Arg Ala 115 120 125 Pro Ile Ala Ala Leu Glu His Gln Lys Arg Val Leu Pro Gln Gln Asn 130 135 140 Leu Lys Trp Val Asp Asn Phe Lys Lys Ala Trp Phe Ile Asp Asn Asp 145 150 155 160 Asp Ala Val Ala Leu Lys Ala Arg Val His Asn Glu Leu Phe Glu Gln 165 170 175 Ala Ala Val Gln Cys Leu Ile Gly Tyr Val Phe Ser Asp Tyr Thr Gln 180 185 190 Val Lys Val Ala Met Ser Pro Leu Trp Lys Ser Val Gln Thr Gly Gln 195 200 205 Gln Ile Ser Arg Val Ile Trp Asp Thr Phe Arg Gln Leu Gly Asp Asp 210 215 220 Ala Ser Ser Tyr Leu Asp Cys Leu Ser Val Leu Glu Glu Tyr Thr Glu 225 230 235 240 Ser Lys Gln Ala Lys Leu Ala Gln Val Gln Pro Ser Phe Val Asn Phe 245 250 255 Asp Val Asn Leu Lys Gly Asp Tyr Gln Gln Ser Arg Leu Lys Val Tyr 260 265 270 Tyr Ala Thr Pro Cys Thr Ala Phe Asp Thr Met Val Gln Val Phe Thr 275 280 285 Leu Gly Gly Arg Leu Lys Gly Pro Glu Val Asp His Ala Ile Glu Cys 290 295 300 Leu Arg Val Leu Trp Pro Ser Val Leu Ala Val Pro Glu Asn His Pro 305 310 315 320 Asp Asp Gln Asp Leu Pro Arg Arg Tyr His Ser Val Ala Val Thr Gln 325 330 335 Phe Asn Phe Glu Leu Trp Pro Gly Ala Lys Leu Pro Val Pro Gln Ile 340 345 350 Tyr Leu Pro Thr Asn Phe Tyr Gly Arg Asp Glu Leu Glu Ile Ala Glu 355 360 365 Gly Leu Glu Gly Phe Phe Lys Thr Leu Gly Trp Ser Glu Pro Phe His 370 375 380 Only Tyr Lys Gln Asn Tyr Ile Only Thr Cys Only Thr Pro Glu Gly Lys 385 390 395 400 Trp Lys Ala Ile Gln His Asp Val Ser Phe Ser Phe Lys Asp Ser Ser 405 410 415 Pro Tyr Val Ser Val Tyr Tyr Lys Pro Glu Leu Ser Val Leu Ser Ser Ser 420 425 430 Pro Ser <210> 31 <211> 1354 <212> DNA <213> Hypoxylon policicidum <400> 31 atggatgctg cttcactct tacgcatgca ccggtatctc agccatggca gtcctagct 60 caagggttgg ggttcgtcaa tgagcatgaa gggtactggt ggtctaagct tggacctcct 120 ctcggtaaaa tgatgaactg ggctcgatac tcgacatcgg aacagtacag agtcctagca 180 ttcctttaca aatatcttct ccctgcctgc ggcccaaagc ctggtgatga tggtgagctg 240 ttctggaagg ttttcatcag ctatgattac acgcccattc agctcagtct caattttcac 300 aatggcaaaa tgacgctgcg caccgcgaac ataccaatta gcgataaatc gggaaccgca 360 420 ccatcccagg atctacgttg gttcaaccac tttgcatccc agtacttctt cgacaaggac 480 acggcagcct ctctaaagac caaggtcgat aagctccgag tccagcaggg agttcagtgt 540 atgctgagcc acgactttcc tgagcgtgat gtccaatgca aagtggcttt ctgcccgctt 600 tggaaagccg tcgctacagg tctttccaac aaggagatca tctgggattc gattctaggg 660 ctcggagatg acatcatccc atacaagcga gcgcttgctg tccttgaaca gtacacatcg 720 tccgaaaatg cagcgaaagc aggagtgcga cctgtattct tcgctttcga tacggtgtta 780 aaagataatt acaagagctc tcgtatcaag atctactacc tcacaacacg gacagccttc 840 aactctatgg tcgacatcta cacacttgga ggcctgctaa aagggcctga tatccaaaaa 900 ggagtagagg cccttgaagt gctctggaaa gccgtcctca acgtccccga ggggtggccc 960 gatgataaag atctacccat gaatccacac cgtgtgcgg cagtaatctt caattttgag 1020 ctgtggccag gagcagagtt tccgagtccc aaggcctatc tcccagccca ttattatggc 1080 cggcctgatt tggagatagc tgatggtatg gactacttct tcaagcagca agggttggat 1140 ggggtatatg gttcttacaa ggaactat ttgaagtgct tgtacgacct cctcacctca 1200 ctcacatcta ccaagctatg gctaatagat cagaagtacg aactcacaga accaactcac 1260 agccctccac catgatattt ctttttcatt caaagggtcc aatgcctacg ttacggtgta 1320 ctacaagccc gagctatctc tagataccga gtag 1354 <210> 32 <211> 1302 <212> DNA <213> Hypoxylon policicidum <400> 32 atggatgctg cttcaactct tacgcatgca ccggtatctc agccatggca gtccctagct 60 caagggttgg ggttcgtcaa tgagcatgaa gggtactggt ggtctaagct tggacctcct 120 ctcggtaaaa tgatgaactg ggctcgatac tcgacatcgg aacagtacag agtcctagca 180 ttcctttaca aatatcttct ccctgcctgc ggcccaaagc ctggtgatga tggtgagctg 240 ttctggaagg ttttcatcag ctatgattac acgcccattc agctcagtct caattttcac 300 aatggcaaaa tgacgctgcg caccgcgaac ataccaatta gcgataaatc gggaaccgca 360 420 ccatcccagg atctacgttg gttcaaccac tttgcatccc agtacttctt cgacaaggac 480 acggcagcct ctctaaagac caaggtcgat aagctccgag tccagcaggg agttcagtgt 540 atgctgagcc acgactttcc tgagcgtgat gtccaatgca aagtggcttt ctgcccgctt 600 tggaaagccg tcgctacagg tctttccaac aaggagatca tctgggattc gattctaggg 660 ctcggagatg acatcatccc atacaagcga gcgcttgctg tccttgaaca gtacacatcg 720 tccgaaaatg cagcgaaagc aggagtgcga cctgtattct tcgctttcga tacggtgtta 780 aaagataatt acaagagctc tcgtatcaag atctactacc tcacaacacg gacagccttc 840 aactctatgg tcgacatcta cacacttgga ggcctgctaa aagggcctga tatccaaaaa 900 ggagtagagg cccttgaagt gctctggaaa gccgtcctca acgtccccga ggggtggccc 960 gatgataaag atctacccat gaatccacac cgttgtgcgg cagtaatctt caattttgag 1020 ctgtggccag gagcagagtt tccgagtccc aaggcctatc tcccagccca ttattatggc 1080 cggcctgatt tggagatagc tgatggtatg gactacttct tcaagcagca agggttggat 1140 ggggtatatg gttcttacaa ggagaactat ttgaagtgct taagtacgaa ctcacagaac 1200 caactcacag ccctccacca tgatatttct ttttcattca aagggtccaa tgcctacgtt 1260 acggtgtact acaagcccga gctatctcta gataccgagt ag 1302 <210> 33 <211> 433 <212> PRT <213> Hypoxylon pulicicidum <400> 33 Met Asp Ala Ala Ser Thr Leu Thr His Ala Pro Val Ser Gln Pro Trp 1 5 10 15 Gln Ser Leu Ala Gln Gly Leu Gly Phe Val Asn Glu His Glu Gly Tyr 20 25 30 Trp Trp Ser Lys Leu Gly Pro Pro Leu Gly Lys Met Met Asn Trp Ala 35 40 45 Arg Tyr Ser Thr Ser Glu Gln Tyr Arg Val Leu Ala Phe Leu Tyr Lys 50 55 60 Tyr Leu Leu Pro Ala Cys Gly Pro Lys Pro Gly Asp Asp Gly Glu Leu 65 70 75 80 Phe Trp Lys Val Phe Ile Ser Tyr Asp Tyr Thr Pro Ile Gln Leu Ser 85 90 95 Leu Asn Phe His Asn Gly Lys Met Thr Leu Arg Thr Ala Asn Ile Pro 100 105 110 Ile Ser Asp Lys Ser Gly Thr Ala Asp Asp Pro Ile Asn Gln Gln Ala 115 120 125 Ser Val Asp Ala Ile Ile Arg Gln Glu Arg Val Leu Pro Ser Gln Asp 130 135 140 Leu Arg Trp Phe Asn His Phe Ala Ser Gln Tyr Phe Phe Asp Lys Asp 145 150 155 160 Thr Ala Ala Ser Leu Lys Thr Lys Val Asp Lys Leu Arg Val Gln Gln 165 170 175 Gly Val Gln Cys Met Leu Ser His Asp Phe Pro Glu Arg Asp Val Gln 180 185 190 Cys Lys Val Ala Phe Cys Pro Leu Trp Lys Ala Val Ala Thr Gly Leu 195 200 205 Ser Asn Lys Glu Ile Ile Trp Asp Ser Ile Leu Gly Leu Gly Asp Asp 210 215 220 Ile Ile Pro Tyr Lys Arg Ala Leu Ala Val Leu Glu Gln Tyr Thr Ser 225 230 235 240 Ser Glu Asn Ala Ala Lys Ala Gly Val Arg Pro Val Phe Phe Ala Phe 245 250 255 Asp Thr Val Leu Lys Asp Asn Tyr Lys Ser Ser Arg Ile Lys Ile Tyr 260 265 270 Tyr Leu Thr Thr Arg Thr Ala Phe Asn Ser Met Val Asp Ile Tyr Thr 275 280 285 Leu Gly Gly Leu Leu Lys Gly Pro Asp Ile Gln Lys Gly Val Glu Ala 290 295 300 Leu Glu Val Leu Trp Lys Ala Val Leu Asn Val Pro Glu Gly Trp Pro 305 310 315 320 Asp Asp Lys Asp Leu Pro Met Asn Pro His Arg Cys Ala Ala Val Ile 325 330 335 Phe Asn Phe Glu Leu Trp Pro Gly Ala Glu Phe Pro Ser Pro Lys Ala 340 345 350 Tyr Leu Pro Ala His Tyr Tyr Gly Arg Pro Asp Leu Glu Ile Ala Asp 355 360 365 Gly Met Asp Tyr Phe Phe Lys Gln Gln Gly Leu Asp Gly Val Tyr Gly 370 375 380 Tyr Lys Glu Asn Tyr Leu Lys Cys Leu Serves Thr Asn Serves Gln Asn 385 390 395 400 Gln Leu Thr Ala Leu His His Asp Ile Ser Phe Ser Phe Lys Gly Ser 405 410 415 Asn Ala Tyr Val Thr Val Tyr Tyr Lys Pro Glu Leu Ser Leu Asp Thr 420 425 430 Glu <210> 34 <211> 1562 <212> DNA <213> Hypoxylon policicidum <400> 34 atggcgcctg atagacttgg tcccgagggc acagcacgcc ccaattccag tggcatctca 60 gttatcgtgg tcggccttgg aattgctgc ccacagctg cattgagtg cccaccgaag 120 ggccattcag tcattgcctt cgagcgaatg aagatgtcg aaccctttgg tgagtttggc 180 ttcgaatccc catattggtt actccgccaa cctagtaaag gcgatagcat catcatcggc 240 agcaacggcg gccgtatttt cggcaaatgg ggccgcggtg aggtacgcaa tgctatgcaa 300 gcctggcgat atacgcctac ccatgccgac atttacgata ccgccggaag attcatggcc 360 cagtctgaga ttcccaaagc tgcggatgac atgtacttca ctcttcgagg cagactagcg 420 aagaccttct acgaacacgc acaaagcctc ggcattgata tgaggatggg gtcgaaagtg 480 actgaatttt gggaagacag caatcgggct ggaatcgttg tggaaggaga gaggtttgag 540 gccgactgtg ttattgcgc tgatggcata cacagcaagt ctcgctcctt gttcacttct 600 ctaaacgctc aaccatttcg ttctggtttc tctattttca gggggaaagc ggacgctaat 660 gcgattattg ccgatcccga tgcgaaatgg atccttgacc agacagagaa caccgatcag 720 ttcaaagtgt ttctggggaa ggagatctgt gttgtcataa ttacctgcgg gtaggccgt 780 cagtggtct cagtgctat catagaggtg agcaatacct atctcccaga taccaataat 840 atataggcagg taggctttgg actatatctt cctgttctgc tcacacaaga ctgccccagg 900 acctaaatga agcggaacag tcgtggtcga cccatgccaa cccggatgat ctattggacg 960 ccatcaaaga ctggccgtgc aggcgccaga tcgaaccaat cgttcggaag ataccgaag 1020 accagttcat cgactatccc cttctaactg tgtctccact ggacacgtgg gtatcccagc 1080 acgggcggat gattctaata ggagatgctg ctcatccatt cttccgact tccggacaag 1140 gaggcgcaca agccatggag gatgcagctg tgcttgcaat ttgcctcgag ttggcaggga 1200 aaggaaacat ccccctggct cttcatgcaa cagaaaagat caggtctagt ttcccggcccc 1260 ttcgggctac tctccctttc cattttatcc gttttcctta cttcgctaa taccacgtaa 1320 cttccagaaa gagccgagct tcagtcctcc aactaaacag gacgtattca gaaggggttc 1380 aactagcacc tgcgctgccg aaatccaaag acagtatgtc tgttccaaat gttccagtaa 1440 tggattggat ctggcatcac tgctgccagt cctacgcata tgatgagttc gacaaggtag 1500 cggaggcgat tcaaagcggg agtgaataca ttccacataa tcttccagaa gatggtacgt 1560 of 1562 <210> 35 <211> 1386 <212> DNA <213> Hypoxylon pulicicidum <400> 35 atggcgcctg atagacttgg tcccgagggc acagcacgcc ccaattccag tggcatctca 60 gttatcgtgg tcggccttgg aattgctggc ctaacagctg caattgagtg ccaccggaag 120 ggccattcag tcattgcctt cgagcgaatg aaggatgtcg aaccctttgg tgagtttggc 180 ttcgaatccc catattggtt actccgccaa cctagtaaag gcgatagcat catcatcggc 240 agcaacggcg gccgtatttt cggcaaatgg ggccgcggtg aggtacgcaa tgctatgcaa 300 gcctggcgat atacgcctac ccatgccgac atttacgata ccgccggaag attcatggcc 360 cagtctgaga ttcccaaagc tgcggatgac atgtacttca ctcttcgagg cagactagcg 420 aagaccttct acgaacacgc acaaagcctc ggcattgata tgaggatggg gtcgaaagtg 480 actgaatttt gggaagacag caatcgggct ggaatcgttg tggaaggaga gaggtttgag 540 gccgactgtg ttatttgcgc tgatggcata cacagcaagt ctcgctcctt gttcacttct 600 ctaaacgctc aaccatttcg ttctggtttc tctattttca gggggaaagc ggacgctaat 660 gcgattattg ccgatcccga tgcgaaatgg atccttgacc agacagagaa caccgatcag 720 ttcaaagtgt ttctggggaa ggagatctgt gttgtcataa ttacctgcgg gtaggccgt 780 cagtggtct cagtgctat catagggac ctaaatgaag cggaacagtc gtggtcgacc 840 catgccaacc cggatgatct attggacgcc atcaaagact ggccgtgcag gcgccagatc 900 gaaccaatcg ttcggaagat atccgaagac cagttcatcg actatcccct tctaactgtg 960 tctccactgg acacgtgggt atcccagcac gggcggatga ttctaatagg agatgctgct 1020 catccattct ttccgacttc cggacaagga ggcgcacaag ccatggagga tgcagctgtg 1080 cttgcaattt gcctcgagtt ggcagggaaa ggaaacatcc ccctggctct tcatgcaaca 1140 gaaaagatca gaaagagccg agcttcagtc ctccaactaa acaggacgta ttcagaaggg 1200 gttcaactag cacctgcgct gccgaaatcc aaagacagta tgtctgttcc aaatgttcca 1260 gtaatggatt ggatctggca tcactgctgc cagtcctacg catatgatga gttcgacaag 1320 gtagcggagg cgattcaaag cgggagtgaa tacattccac ataatcttcc agaagatggt 1380 acgtag 1386 <210> 36 <211> 461 <212> PRT <213> Hypoxylon pulicicidum <400> 36 Met Ala Pro Asp Arg Leu Gly Pro Glu Gly Thr Ala Arg Pro Asn Ser 1 5 10 15 Ser Gly Ile Ser Val Ile Val Val Gly Leu Gly Ile Ala Gly Leu Thr 20 25 30 Ala Ala Ile Glu Cys His Arg Lys Gly His Ser Val Ile Ala Phe Glu 35 40 45 Arg Met Lys Asp Val Glu Pro Phe Gly Glu Phe Gly Phe Glu Ser Pro 50 55 60 Tyr Trp Leu Leu Arg Gln Pro Ser Lys Gly Asp Ser Ile Ile Ile Gly 65 70 75 80 Ser Asn Gly Gly Arg Ile Phe Gly Lys Trp Gly Arg Gly Glu Val Arg 85 90 95 Asn Ala Met Gln Ala Trp Arg Tyr Thr Pro Thr His Ala Asp Ile Tyr 100 105 110 Asp Thr Ala Gly Arg Phe Met Ala Gln Ser Glu Ile Pro Lys Ala Ala 115 120 125 Asp Asp Met Tyr Phe Thr Leu Arg Gly Arg Leu Ala Lys Thr Phe Tyr 130 135 140 Glu His Ala Gln Ser Leu Gly Ile Asp Met Arg Met Gly Ser Lys Val 145 150 155 160 Thr Glu Phe Trp Glu Asp Ser Asn Arg Ala Gly Ile Val Val Glu Gly 165 170 175 Glu Arg Phe Glu Ala Asp Cys Val Ile Cys Ala Asp Gly Ile His Ser 180 185 190 Lys Ser Arg Ser Leu Phe Thr Ser Leu Asn Ala Gln Pro Phe Arg Ser 195 200 205 Gly Phe Ser Ile Phe Arg Gly Lys Ala Asp Ala Asn Ala Ile Ile Ala 210 215 220 Asp Pro Asp Ala Lys Trp Ile Leu Asp Gln Thr Glu Asn Thr Asp Gln 225 230 235 240 Phe Lys Val Phe Leu Gly Lys Glu Ile Cys Val Val Ile Ile Thr Cys 245 250 255 Gly Leu Gly Arg Ala Val Val Cys Ser Ala Met His Arg Asp Leu Asn 260 265 270 Glu Ala Glu Gln Ser Trp Ser Thr His Ala Asn Pro Asp Asp Leu Leu 275 280 285 Asp Ala Ile Lys Asp Trp Pro Cys Arg Arg Gln Ile Glu Pro Ile Val 290 295 300 Arg Lys Ile Ser Glu Asp Gln Phe Ile Asp Tyr Pro Leu Leu Thr Val 305 310 315 320 Ser Pro Leu Asp Thr Trp Val Ser Gln His Gly Arg Met Ile Leu Ile 325 330 335 Gly Asp Ala Ala His Pro Phe Phe Pro Thr Ser Gly Gln Gly Gly Ala 340 345 350 Gln Ala Met Glu Asp Ala Ala Val Leu Ala Ile Cys Leu Glu Leu Ala 355 360 365 Gly Lys Gly Asn Ile Pro Leu Ala Leu His Ala Thr Glu Lys Ile Arg 370 375 380 Lys Ser Arg Ala Ser Val Leu Gln Leu Asn Arg Thr Tyr Ser Glu Gly 385 390 395 400 Val Gln Leu Ala Pro Ala Leu Pro Lys Ser Lys Asp Ser Met Ser Val 405 410 415 Pro Asn Val Pro Val Met Asp Trp Ile Trp His His Cys Cys Gln Ser 420 425 430 Tyr Ala Tyr Asp Glu Phe Asp Lys Val Ala Glu Ala Ile Gln Ser Gly 435 440 445 Ser Glu Tyr Ile Pro His Asn Leu Pro Glu Asp Gly Thr 450 455 460 <210> 37 <211> 1844 <212> DNA <213> Hypoxylon policicidum <400> 37 atgataacag catccacttc cgtgtttggt ggcttaatcc ttgcctttat cttctcactt 60 ttatataaga ataagaagac ccgtatccca gcagaaatcg accgcgtaag gacgggaggt 120 ttcctagcac acattagagc ctttgggtgc aggttaggta cgcgagttga tgatattcgc 180 aatggatata ataaggttag ttgctcctat agctgctcac aaaatact caacacatga 240 tatctcaata cgcagttcaa caaaaacggt aagccgtttg tgatccagga ttctacctttt 300 atcccacagg tcgtcatacc acctcaatac ctgggatggt tgaaggagca accagagaag 360 gctctttctg cagaaaccgt gaggctagaa cagcttggac ttcgctattt ggtccctagc 420 tcagatcccg agatggtcca tttgttgaca gacgtcgtgt gtcgctatct tacccgcaat 480 tttcaaagag tacaagagcg tttatatgag gagctccata tgagtacgga tgaaatcatg 540 gggctggagg caactgagtg gcgtcagatt tgtctccatg aagcgatgga aacgattctt 600 cgaaggatga ttagttgcgt cctgattggt ctcccatggt gtcgagatga ggaatgctta 660 aaatcgtgga ccggatttct tcactgcatg gccattgcag gaactattct aggggcagta 720 acaccttggt tcctacggcc actcctcgga ctactgctca agccgcccgt tggctatatg 780 cgcagaagat cactgcgtta tcttactccg atctttactg aaagatggaa aaaaattgag 840 aagcatgaga agagctcact gacgacgcgt gagctgccgg atgacttcgt aacatggtgt 900 attcaggagg tacggaatgg taatatgaaa gaaaaacacg atgctcttag ccttgcgtcg 960 gaatttctat tctttgtgag tcactctacg cgttggtatc atggtttccc atattcaata 1020 cgcgatgcga tggatcgagg ctctaacgag aacgcagtcg atcgcattct ttgacgcgcc 1080 tataggagct gcagaagtca ccatgctcga tcttctaagt gcggatccca caataggtta 1140 ctgggaaaag cttgtggaag aagccactac tgcattcaga acagacgagg actggattca 1200 tgcaggcact gtgtcaaaat tggcatacac ggacagcgca atcagggagt ctctacgccg 1260 aaaccctttc agcatccgaa acgtgacccg agggtgata ggaaaagatg ggctaacact gccatccggt acgcgtctgc cacagggcac ttggatcacg accgctctcg ccaatataca 1380 1440. tcatgatgca agattttact caaatcccac cgaataccag cctttccgtt ttgtggccag agacgcgttt cacacagagg ggaaagaggg tagcgaaaag gttttacagc cctctgaagc tatcttgacg agcacaattg atgaaaggct cttgacattc ggatatgggc gtcgagcatg gtacgaaatc caactgtctc ctagagtctc aaactcttat atcctgtctc cagtcgaacc tacctaggta ctaatgcttt tgcaattact gtagccccgg ccgatggttc gcttcacaca tactgaaaat gttgatcgcg fathercacga tcaattatga catacagccc ttgacgggac cgccgaaaaa agtcaaattc gcagatttca ccgtgccatc gccgagtatc aaaatcattg tgcgccgga gaatctcgct taccttaggc aacgtgagcg ttga <210> 38 <211> 1524 <212> DNA <213> Hypoxylon pulcicidum <400> 38 atgataacag catccacttc cgtgtttggt ggcttaatcc ttgcctttat cttctcactt 60 ttatataaga ataagaagac ccgtatccca gcagaaatcg accgcgtaag gacgggaggt 120 ttcctagcac acattagagc ctttgggtgc aggttaggta cgcgagttga tgatattcgc 180 aatggatata ataagttcaa caaaaacggt aagccgtttg tgatccagga ttctaccttt 240 atcccacagg tcgtcatacc acctcaatac ctgggatggt tgaaggagca accagagaag 300 gctctttctg cagaaaccgt gaggctagaa cagcttggac ttcgctattt ggtccctagc 360 tcagatcccg agatggtcca tttgttgaca gacgtcgtgt gtcgctatct tacccgcaat 420 tttcaaagag tacaagagcg tttatatgag gagctccata tgagtacgga tgaaatcatg 480 gggctggagg caactgagtg gcgtcagatt tgtctccatg aagcgatgga aacgattctt 540 cgaaggatga ttagttgcgt cctgattggt ctcccatggt gtcgagatga ggaatgctta 600 aaatcgtgga ccggatttct tcactgcatg gccattgcag gaactattct aggggcagta 660 acaccttggt tcctacggcc actcctcgga ctactgctca agccgcccgt tggctatatg 720 cgcagaagat cactgcgtta tcttactccg atctttactg aaagatggaa aaaaattgag 780 aagcatgaga agagctcact gacgacgcgt gagctgccgg atgacttcgt aacatggtgt 840 attcaggagg tacggaatgg agctgcagaa gtcaccatgc tcgatcttct aagtgcggat 900 cccacaatag gttactggga aaagcttgtg gaagaagcca ctactgcatt cagaacagac 960 gaggactgga ttcatgcagg cactgtgtca aaattggcat acacggacag cgcaatcagg 1020 gagtctctac gccgaaaccc tttcagcatc cgaaacgtga cccgagaggt gataggaaaa 1080 gatgggctaa cactgccatc cggtacgcgt ctgccacagg gcacttggat cacgaccgct 1140 ctcgccaata tacatcatga tgcaagattt tactcaaatc ccaccgaata ccagcctttc 1200 cgttttgtgg ccagagacgc gtttcacaca gaggggaaag agggtagcga aaaggtttta 1260 cagccctctg aagctatctt gacgagcaca attgatgaaa ggctcttgac attcggatat 1320 gggcgtcgag catgccccgg ccgatggttc gcttcacaca tactgaaaat gttgatcgcg 1380 tatatcacga tcaattatga catacagccc ttgacgggac cgccgaaaaa agtcaaattc 1440 gcagatttca ccgtgccatc gccgagtatc aaaatcattg tgcgccggaa gaatctcgct 1500 taccttaggc aacgtgagcg ttga 1524 <210> 39 <211> 507 <212> PRT <213> Hypoxylon pulicicidum <400> 39 Met Ile Thr Ala Ser Thr Ser Val Phe Gly Gly Leu Ile Leu Ala Phe 1 5 10 15 Ile Phe Ser Leu Leu Tyr Lys Asn Lys Lys Thr Arg Ile Pro Ala Glu 20 25 30 Ile Asp Arg Val Arg Thr Gly Gly Phe Leu Ala His Ile Arg Ala Phe 35 40 45 Gly Cys Arg Leu Gly Thr Arg Val Asp Asp Ile Arg Asn Gly Tyr Asn 50 55 60 Lys Phe Asn Lys Asn Gly Lys Pro Phe Val Ile Gln Asp Ser Thr Phe 65 70 75 80 Ile Pro Gln Val Val Ile Pro Pro Gln Tyr Leu Gly Trp Leu Lys Glu 85 90 95 Gln Pro Glu Lys Ala Leu Ser Ala Glu Thr Val Arg Leu Glu Gln Leu 100 105 110 Gly Leu Arg Tyr Leu Val Pro Ser Ser Asp Pro Glu Met Val His Leu 115 120 125 Leu Thr Asp Val Val Cys Arg Tyr Leu Thr Arg Asn Phe Gln Arg Val 130 135 140 Gln Glu Arg Leu Tyr Glu Glu Leu His Met Ser Thr Asp Glu Ile Met 145 150 155 160 Gly Leu Glu Ala Thr Glu Trp Arg Gln Ile Cys Leu His Glu Ala Met 165 170 175 Glu Thr Ile Leu Arg Arg Met Ile Ser Cys Val Leu Ile Gly Leu Pro 180 185 190 Trp Cys Arg Asp Glu Glu Cys Leu Lys Ser Trp Thr Gly Phe Leu His 195 200 205 Cys Met Ala Ile Ala Gly Thr Ile Leu Gly Ala Val Thr Pro Trp Phe 210 215 220 Leu Arg Pro Leu Leu Gly Leu Leu Leu Lys Pro Pro Val Gly Tyr Met 225 230 235 240 Arg Arg Arg Ser Leu Arg Tyr Leu Thr Pro Ile Phe Thr Glu Arg Trp 245 250 255 Lys Lys Ile Glu Lys His Glu Lys Ser Ser Leu Thr Thr Arg Glu Leu 260 265 270 Pro Asp Asp Phe Val Thr Trp Cys Ile Gln Glu Val Arg Asn Gly Ala 275 280 285 Ala Glu Val Thr Met Leu Asp Leu Leu Ser Ala Asp Pro Thr Ile Gly 290 295 300 Tyr Trp Glu Lys Leu Val Glu Glu Ala Thr Thr Ala Phe Arg Thr Asp 305 310 315 320 Glu Asp Trp Ile His Ala Gly Thr Val Ser Lys Leu Ala Tyr Thr Asp 325 330 335 Ser Ala Ile Arg Glu Ser Leu Arg Arg Asn Pro Phe Ser Ile Arg Asn 340 345 350 Val Thr Arg Glu Val Ile Gly Lys Asp Gly Leu Thr Leu Pro Ser Gly 355 360 365 Thr Arg Leu Pro Gln Gly Thr Trp Ile Thr Thr Ala Leu Ala Asn Ile 370 375 380 His His Asp Ala Arg Phe Tyr Ser Asn Pro Thr Glu Tyr Gln Pro Phe 385 390 395 400 Arg Phe Val Ala Arg Asp Ala Phe His Thr Glu Gly Lys Glu Gly Ser 405 410 415 Glu Lys Val Leu Gln Pro Ser Glu Ala Ile Leu Thr Ser Thr Ile Asp 420 425 430 Glu Arg Leu Leu Thr Phe Gly Tyr Gly Arg Ala Cys Pro Gly Arg 435 440 445 Trp Phe Can Serve His There Are Lys And There Are Tyr There Thr There 450 455 460 Asn Tyr Asp Ile Gln Pro Leu Thr Gly Pro Pro Lys Lys Val Lys Phe 465 470 475 480 Ala Asp Phe Thr Val Pro Ser Pro Ser Ile Lys Ile Ile Val Arg Arg 485,490,495 Lys Asn Leu Ala Tyr Leu Arg Gln Arg Glu Arg 500,505 <210> 40 <211> 1294 <212> DNA <213> Penicillium paxilli <400> 40 ttaaactctt cctttctcat tagtaggta ctccacttca agtagatcga gatcttatg 60 aattccagga ttggcccta aggaagtctc cagctcttt acatagccac gagcctccgc 120 actaagtcga cttagggtct ccctagtata ttggaagctc cccgtcgact ccatgatctt 180 cacagcccgg attttgactg ctcgtcctc tgtgcgttgc tttagaatat cgagaagctc 240 ggagctctct ggagatgcac ggatgctgtg tatgatagga taggaact tcccctccgt 300 caaatcttcc atcagccctt tttctcagc atagagtccg ctctgcagat tcatatagtc 360 atcacgaatt tggaataa cacccaatag ctcgacaagt ttagaaagt cactgtaaaa 420 tactaattag cattgctgcg aagtgaccgt tatggattag aaacttacgt attcttgcgg 480 gattggatac gcatcaatc gaggggccagg ttgaaagac ctccagtttt gtacatacc 540 atccgggtgt attcctccctc ggtcggacaa accaccatat cacgccaata taggtccatg 600 ccctggccaa gatgtagatc tagcagtgac cttgtaaata ttcgaggc gcggggggtct 660 ccaatctctt tcaatcttgc tgttggaga tagtggcat agttggctga gttgatgtt 720 tgtgctacac cgtatacatc atgagccaca ggctttcctc tccggagccg tgatgcatct 780 tgaatatcat cgatactata atcaccatga tgttagaata gaacaggagc gctggggttg 840 gaaatctcac agaaggtg cagtgtgaag tagttttatt atatctttca caatagacaa 900 cttctcctct ggaagctgga gccatatgtt gaaggagtcg atcagcttgc ttcggatgtc 960 ttttcccgga atcgctagaa gatagtctag aggtccacga acaatctaag aagaacaaac gattggtata atgaaacaca aggggaatcc caagagcaat gacagaactg accttgttat actgaatagc aatatcgtca acgtcatctt ctagcacctg cactgttcta atcgtgcttg gtgctttgct ggaatcactc aggagtcttg ggaatccttg gaagtttgat tcccagtaat 1260. the father of the father of the father of the father of the father of the father of the cctcgacga cgaagttcag agcttctgca aggatgtagg acat <210> 41 <211> 1116 <212> DNA <213> Penicillium paxilli <400> 41 ttaaactctt cctttctcat tagged ctccacttca tagged gaatcttatg aattccagga ttgggcccta aggaagtctc cagcttcttt acatagccac gagcctccgc actaagtcga cttagggtct ccctagtata ttggaagctc cccgtcgact ccatgatctt cacagcccgg attttgactg cttcgtcctc tgtgcgttgc tttagaatat cgagaagctc ggagctctct ggagatgcac ggatgctgtg tatgatagga taggagaact tcccctccgt 300 caaatcttcc atcagccctt tttctcagc atagagtccg ctctgcagat tcatatagtc 360 atcacgaatt tggaatataa cacccaatag ctcgacaagt ttagaaaagt cagtattctt 420 gcgggattgg atacgcatca aatcgagggc caggttgaaa agacctccag ttttgtacat 480 aaccatccgg gtgtattcct cctcggtcgg acaaaccacc atatcacgcc atataggtc 540 catgccctgg ccaagatgta gatctagcag tgaccttgta aatatttcga aggcgcgggg 600 gtctccaatc tctttcaatc ttgcttgttg gagatagtag gcatagttgg ctgagttgat 660 agttgtgct acaccgtata catcatgagc cacaggcttt cctctccgga gccgtgatgc 720 atcttgaata tcatcgataa gaagggatgc agtgtgaagt aagtttatta tatctttcac 780 aatagacaac ttctcctctg gaagctggag ccatatgttg aaggagtcga tcagcttgct 840 tcggatgtct tttcccggaa tcgctagaag atagtctaga ggtccacgaa caatcttgtt 900 atactgaata gcaatatcgt caacgtcatc ttctagcacc tgcactgttc taatcgtgct 960 tggtgctttg ctggaatcac tcaggagtct tgggaatcct tggaagtttg attcccagta 1020 attatcagca gataatgagt gtgaagctcc ccaataatta agatgagata ttcctcgacg 1080 aacgaagttc agagcttctg caaggatgta ggacat 1116 <210> 42 <211> 1077 <212> DNA <213> Penicillium paxilli <400> 42 atgggcgtag cagggagcgg agttctttac tttcttttca acaatgtccc gagtcctcgc 60 ttctggttga agaaaaccca gttgatagga accgagaacc cagaaggcat aactggctac 120 gaatgccctt atgaatatct gcggaagtca tacggcaagc atcactgggc agcgttcgtt 180 gacaagctat cgcccaacct tcaaaatgag gatccagcta aataccgcat ggtgcttgaa 240 acaatggatg tcattcacct atgcttgatg atggtcgacg atgtaagatc atttgccatt 300 atatagaggc tttattatca ttgtatctaa cttctgattc ctagatctcc gatggaagcg 360 aatatcgcaa aggaaagccg gctgcgcaca aaatttatgg tgcgcctgaa acagcgaatc 420 gggcttatta tcgagttaca caaatattgg ctcaaactgc gacagaattc ccacgtcttt 480 cgccttggtt gatgactgat cttcgggata ttctcgaggg tcaagatatg tcccttgtct 540 ggcgccgaga cggagtcaat gggttccctg gaactgcatc ggagagaact gctgcttaca 600 ggcgccgaga cggagtcaat gggttccctg gaactgcatc ggagagaact gctgcttaca 600 agcgcatggt tctgctaaag acaggtggac tatttcgtct actcgggcat ctcactctcg 660 agcgcatggt tctgctaaag acaggtggac tatttcgtct actcgggcat ctcactctcg 660 agaacaattc catggatgaa gcctttagca cccttgggta agaaagaaaa atattttcgg 720 agaacaattc catggatgaa gcctttagca cccttgggta agaaagaaaa atattttcgg 720 cagccttctg aattttgcta atatttaatg attcctagct ggcattcgca attgcaaaat 780 cagccttctg aattttgcta atatttaatg attcctagct ggcattcgca attgcaaaat 780 gactgcaaga atgtctactc gtcggaatat gccaagatga agggcgttgt agcagaagat 840 gactgcaaga atgtctactc gtcggaatat gccaagatga agggcgttgt agcagaagat 840 ttgctcaatc gtgagatgac ataccccatc gtactcgcac tggacgcctc tggtggtcat 900 ttgctcaatc gtgagatgac ataccccatc gtactcgcac tggacgcctc tggtggtcat tgggtagagg cagctctaaa gtcgccctct cggcgaaacg tcggaaatgc cttgaagata 960 tgggtagagg cagctctaaa gtcgccctct cggcgaaacg tcggaaatgc cttgaagata 960 atacagtgcg actatgttcg agatgtttgc atggcagagc tggcgagatc tggtgccccg 1020 atacagtgcg actatgttcg agatgtttgc atggcagagc tggcgagatc tggtgccccg 1020 gttaaggaat ggttgaagtt atggaaacgg gaggagaagc ttgacctgaa ggcatga <210> 43 <211> 954 <212> DNA <213> Penicillium paxilli <400> 43 atgggcgtag cagggagcgg agttctttac tttcttttca acaatgtccc gagtcctcgc 60 atgggcgtag cagggagcgg agttctttac tttcttttca acaatgtccc gagtcctcgc 60 ttctggttga agaaaaccca gttgatagga accgagaacc cagaaggcat aactggctac 120 gaatgccctt atgaatatct gcggaagtca tacggcaagc atcactgggc agcgttcgtt 180 gacaagctat cgcccaacct tcaaaatgag gatccagcta aataccgcat ggtgcttgaa 240 acaatggatg tcattcacct atgcttgatg atggtcgacg atactccga tggaagcgaa 300 tatcgcaaag gaaagccggc tgcgcacaaa attatggtg cgcctgaaac agcgaatcgg 360 gcttattatc gagttacaca aatattggct caaactgcga cagaattccc acgtctttcg 420 ccttggttga tgactgatct tcgggatatt ctcgagggtc aagatatgtc ccttgtctgg 480 cgccgagacg gagtcaatgg gttccctgga actgcatcgg agagaactgc tgcttacaag 540 cgcatggttc tgctaaagac aggtggacta tttcgtctac tcgggcatct cactctcgag 600 aacaattcca tggatgaagc ctttagcacc cttggctggc attcgcaatt gcaaaatgac 660 tgcaagaatg tctactcgtc ggaatatgcc aagatgaagg gcgttgtagc agaagatttg 720 ctcaatcgtg agatgacata ccccatcgta ctcgcactgg acgcctctgg tggtcattgg 780 gtagaggcag ctctaaagtc gccctcgg cgaaacgtcg gaatgcctt gaagatatata 840 cagtgcgact atgttcgaga tgttgcatg gcagagctgg cgagatctgg tgccccggtt 900 aaggaatggt tgaagttag gaacgggag gagaagcttg acctgaaggc atga 954 <210> 44 <211> 317 <212> PRT <213> Penicillium paxilli <400> 44 Met Gly Val Ala Gly Ser Gly Val Leu Tyr Phe Leu Phe Asn Asn Val 1 5 10 15 Pro Ser Pro Arg Phe Trp Leu Lys Lys Thr Gln Leu Ile Gly Thr Glu 20 25 30 Asn Pro Glu Gly Ile Thr Gly Tyr Glu Cys Pro Tyr Glu Tyr Leu Arg 35 40 45 Lys Ser Tyr Gly Lys His Trp Ala Ala Phe Val Asp Lys Leu Ser 50 55 60 Pro Asn Leu Gln Asn Glu Asp Pro Ala Lys Tyr Arg Met Val Leu Glu 65 70 75 80 Thr Met Asp Will Ile His Leu Cys Leu Met Met Will Asp Ile Ser 85 90 95 Asp Gly Ser Glu Tyr Arg Lys Gly Lys Pro Ala Ala His Lys Ile Tyr 100 105 110 Gly Ala Pro Glu Thr Ala Asn Arg Ala Tyr Tyr Arg Val Thr Gln Ile 115 120 125 Leu Ala Gln Thr Ala Thr Glu Phe Pro Arg Leu Ser Pro Trp Leu Met 130 135 140 Thr Asp Leu Arg Asp Ile Leu Glu Gly Gln Asp Met Ser Leu Val Trp 145 150 155 160 Arg Arg Asp Gly Val Asn Gly Phe Pro Gly Thr Ala Ser Glu Arg Thr 165 170 175 Ala Ala Tyr Lys Arg Met Val Leu Leu Lys Thr Gly Gly Leu Phe Arg 180 185 190 Leu Leu Gly His Leu Thr Leu Glu Asn Asn Ser Met Asp Glu Ala Phe 195 200 205 Ser Thr Leu Gly Trp His Ser Gln Leu Gln Asn Asp Cys Lys Asn Val 210 215 220 Tyr Ser Ser Glu Tyr Ala Lys Met Lys Gly Val Val Ala Glu Asp Leu 225 230 235 240 Leu Asn Arg Glu Met Thr Tyr Pro Ile Val Leu Ala Leu Asp Ala Ser 245 250 255 Gly Gly His Trp Val Glu Ala Ala Leu Lys Ser Pro Ser Arg Arg Asn 260 265 270 Val Gly Asn Ala Leu Lys Ile Ile Gln Cys Asp Tyr Val Arg Asp Val 275 280 285 Cys Met Ala Glu Leu Ala Arg Ser Gly Ala Pro Val Lys Glu Trp Leu 290 295 300 Lys Leu Trp Lys Arg Glu Glu Lys Leu Asp Leu Lys Ala 305 310 315 <210> | 45 <211> | 1559 <212> | DNA <213> | Penicillium paxilli <400> | 45 ttaaacttga agaaaataaa acttcagggc accctcgagg tcgaacatca ttgtaagaga 60 ggtatagaca atggttagtc caaatagata gatcataagt tgagtcaaat aactccagct 120 tcgcccctgt ttgctatact tctcccatcc accaccactt cgctttggcg tagggaggaa 180 atcacacatt gtgccatcag caatcgtttt tgacgccata tcagcaggca ggtcaccagc 240 gtatggtgcc caataccggc ttagcaggct atagattatg ccatcacgaa cctgaaaacg 300 (注:对于<210>、<211>、<212>、<213>、<400>等标签,由于不清楚其具体含义和翻译要求,保留原样并添加了竖线“|”以体现与其他文本的区分,你可根据实际情况调整。)aaccaagaat cgagagctct ggtagtagt attgacgcgc tcatatcgaa ggtcgcgata tttttgcaaa aggaactcca tttgtgagga ggtgggaag tatggtccag aggaaatccg catctttcga aggagatttg ccagagcagc ggcatcctca atggccatat tagccccttg 480 accaacattg ggagtcattt tgtggacact gtctcctaag agcacgcagc ggccatggtg 540. ccagactttg aatgtatttt cttccagagc agtcattgaa gatgtctctc ttttgtccca gagctcgcca aaagtaatgt tttcgtaaaa cttgacatcc ctgatctctt ctgccgcaat agatgtttca tgggacgtgt agcgggggct gtcaggatac acgtatttct tgcccagctt 720 ttgaatgacg aaccaata tgcgaccgtc ttttccatgg attgtgacaa tagtgagacc atcgaataaa gcgttgactt gctcgccaag ttttaatccc ggcatcgccg atgaaatgcc 900. cgaaatacag cgaaactcga ccgtaagctc ttcgtacttg ttagcatgaa tgcaaagaca tgaggcctgt tgatgctatc cactcactgg aactatcttg tttgatcttt gatacccgtc 960 tggcaattcc ccttgccttc catatctctc tgcggacgat gctgtggact ccatctgcgc 1020 cgacaagaag atccccacga taaacgtgtc cagttgtggt ggtaattagc actccatcat 1080 ccaatgattc aattgatgta actctttggc ccagacgtat tttgctggga tctggatacc 1140 ctttgtagag aatttcgagc atcttctgtc gatctagaaa cgctatagga aaaccgaacc 1200 tgttgcctca cgggtgccat tttagatgat aatgcaaaac tgtataagat caagataatt 1260 actgaccttt gatcgatgat ctttggatat gagctgctga agttgaaccc atcaggaagc 1320 ccaattgtag ctttgcttag cggttcgata tgctcttcaa cttgatcata gagctgaagc 1380 tgatccagca cgcgagctcc attcggcaga atgccgatgg atgctccaat ctgtggtgct 1440 ggatcgctgg ccttttccag gacaacatgt tttattcccg cacgatgtag acaatgtgcc 1500 aatgtcaatc ctccgatcga cccgcccaca atgataactt gaaactcggc cttttccat 1559 <210> 46 <211> 1434 <212> DNA <213> Penicillium paxilli <400> 46 ttaaacttga agaaaataaa acttcagggc accctcgagg tcgaacatca ttgtaagaga 60 ggtatagaca atggttagtc caaatagata gatcataagt tgagtcaaat aactccagct 120 tcgcccctgt ttgctatact tctcccatcc accaccactt cgctttggcg tagggaggaa 180 atcacacatt gtgccatcag caatcgtttt tgacgccata tcagcaggca ggtcaccagc 240 gtatggtgcc caataccggc ttagcaggct atagattatg ccatcacgaa cctgaaaacg 300 aaccaagaat cgagagctct ggtagatagt attgacgcgc tcatatcgaa ggtcgcgata 360 tttttgcaaa aggaactcca tttgtgagga ggtgggaaag tatggtccag aggaaatccg 420 catctttcga aggagatttg ccagagcagc ggcatcctca atggccatat tagccccttg 480 accaacattg ggagtcattt tgtggacact gtctcctaag agcacgcagc ggccatggtg 540 ccagactttg aatgtatttt cttccagagc agtcattgaa gatgtctctc ttttgtccca 600 gagctcgcca aaagtaatgt tttcgtaaaa cttgacatcc ctgatctctt ctgccgcaat 660 agatgtttca tgggacgtgt agcgggggct gtcaggatac acgtatttct tgcccagctt 720 ttgaatgacg aaccaatata tgcgaccgtc ttttccatgg attgtgacaa tagtgagacc 780 atcgaataaa gcgttgactt gctcgccaag ttttaatccc ggcatcgccg atgaaatgcc 840 gaaaatacag cgaaactcga ccgtaagctt ggaactatct tgtttgatct ttgatacccg 900 tctggcaatt ccccttgcct tccatatctc tctgcggacg atgctgtgga ctccatctgc 960 gccgacaaga agatccccac gataaacgtg tccagttgtg gtggtaatta gcactccatc 1020 atccaatgat tcaattgatg taactctttg gcccagacgt attttgctgg gatctggata 1080 ccctttgtag agaatttcga gcatcttctg tcgatctaga aacgctatag gaaaaccgaa 1140 cctttgatcg atgatctttg gatatgagct gctgaagttg aacccatcag gaagcccaat 1200 tgtagctttg cttagcggtt cgatatgctc ttcaacttga tcatagagct gaagctgatc 1260 cagcacgcga gctccattcg gcagaatgcc gatggatgct ccaatctgtg gtgctggatc 1320 gctggccttt tccaggacaa catgttttat tcccgcacga tgtagacaat gtgccaatgt 1380 caatcctccg atcgacccgc ccacaatgat aacttgaaac tcggcctttt ccat 1434 <210> 47 <211> 477 <212> PRT <213> Penicillium paxilli <400> 47 Met Glu Lys Ala Glu Phe Gln Val Ile Ile Val Gly Gly Ser Ile Gly 1 5 10 15 Gly Leu Thr Leu Ala His Cys Leu His Arg Ala Gly Ile Lys His Val 20 25 30 Val Leu Glu Lys Ala Ser Asp Pro Ala Pro Gln Ile Gly Ala Ser Ile 35 40 45 Gly Ile Leu Pro Asn Gly Ala Arg Val Leu Asp Gln Leu Gln Leu Tyr 50 55 60 Asp Gln Val Glu Glu His Ile Glu Pro Leu Ser Lys Ala Thr Ile Gly 65 70 75 80 Leu Pro Asp Gly Phe Asn Phe Ser Ser Ser Tyr Pro Lys Ile Ile Asp 85 90 95 Gln Arg Phe Gly Phe Pro Ile Ala Phe Leu Asp Arg Gln Lys Met Leu 100 105 110 Glu Ile Leu Tyr Lys Gly Tyr Pro Asp Pro Ser Lys Ile Arg Leu Gly 115 120 125 Gln Arg Val Thr Ser Ile Glu Ser Leu Asp Asp Gly Val Leu Ile Thr 130 135 140 Thr Thr Thr Gly His Val Tyr Arg Gly Asp Leu Leu Val Gly Ala Asp 145 150 155 160 Gly Val His Ser Ile Val Arg Arg Glu Ile Trp Lys Ala Arg Gly Ile 165 170 175 Ala Arg Arg Val Ser Lys Ile Lys Gln Asp Ser Ser Lys Leu Thr Val 180 185 190 Glu Phe Arg Cys Ile Phe Gly Ile Ser Ser Ala Met Pro Gly Leu Lys 195 200 205 Leu Gly Glu Gln Val Asn Ala Leu Phe Asp Gly Leu Thr Ile Val Thr 210 215 220 Ile His Gly Lys Asp Gly Arg Ile Tyr Trp Phe Val Ile Gln Lys Leu 225 230 235 240 Gly Lys Lys Tyr Val Tyr Pro Asp Ser Pro Arg Tyr Thr Ser His Glu 245 250 255 Thr Ser Ile Ala Ala Glu Glu Ile Arg Asp Val Lys Phe Tyr Glu Asn 260 265 270 Ile Thr Phe Gly Glu Leu Trp Asp Lys Arg Glu Thr Ser Ser Met Thr 275 280 285 Ala Leu Glu Glu Asn Thr Phe Lys Val Trp His His Gly Arg Cys Val 290 295 300 Leu Leu Gly Asp Ser Val His Lys Met Thr Pro Asn Val Gly Gln Gly 305 310 315 320 Ala Asn Met Ala Ile Glu Asp Ala Ala Ala Leu Ala Asn Leu Leu Arg 325 330 335 Lys Met Arg Ile Ser Ser Gly Pro Tyr Phe Pro Thr Ser Ser Gln Met 340 345 350 Glu Phe Leu Leu Gln Lys Tyr Arg Asp Leu Arg Tyr Glu Arg Val Asn 355 360 365 Thr Ile Tyr Gln Ser Ser Arg Phe Leu Val Arg Phe Gln Val Arg Asp 370 375 380 Gly Ile Ile Tyr Ser Leu Leu Ser Arg Tyr Trp Ala Pro Tyr Ala Gly 385 390 395 400 Asp Leu Pro Ala Asp Met Ala Ser Lys Thr Ile Ala Asp Gly Thr Met 405 410 415 Cys Asp Phe Leu Pro Thr Pro Lys Arg Ser Gly Gly Gly Trp Glu Lys 420 425 430 Tyr Ser Lys Gln Gly Arg Ser Trp Ser Tyr Leu Thr Gln Leu Met Ile 435 440 445 Tyr Leu Phe Gly Leu Thr Ile Val Tyr Thr Ser Leu Thr Met Met Phe 450 455 460 Asp Leu Glu Gly Ala Leu Lys Phe Tyr Phe Leu Gln Val 465 470 475 <210> 48 <211> 1681 <212> DNA <213> Hypoxylon policicidum <400> 48 atggacaagc taccacagga aatcgtggac atcatcgtct tctttgccaa ctccaacgag 60 caagggaacc aatatcacat gcacggcccc gatgctaagc atcttgcgag acaattagtc 120 actgtttcca agaaattcca gcgcgccatc gagtgtgata cattcaaggt acttcatctc 180 gacttggacc gggctcgtga tgctcgtgat atcctttccc ggttctctag atggtcgcat 240 ctgagactac tagaatttac tgctatcctg cccgactatg acgaagttgc gtgcactcga 300 tatgagaacg ctgcagagaa agcagagaat tcgcgggtgt ttacggaaaa catcacagag 360 ttgttccacc ttctctctca atgcccagtt gcatgtgctt ctagacctgt ggtattgagg 420 gtagagatat attccaagac ggatcgcatc tgtgtcagcc acagtcttca aggccgtgtg 480 actcaaaagg agacgcacgc caagccagtg tctggtgaca taaggaggtg gaggtggaag 540 cggtcaaaac tacagcttga caagtcagtt cgtcttgcct caatcaaaca cctaaatata 600 cctgtcttcc agatgcaaag gcgcccgtta ttattccgac atgttgctgc tgatagcatt 660 atgcgattaa tatcagcaat gcctccctcc ctcaagttta ccgtcaaccg gttttcagat 720 accgaaaaga acgatcttgc tcatcgaatt gcacatcgtg agggtaagcg ttagggtctc 780 accattgatc gattaatatt cccacccatg cgttctctaa actaacatat ttccagatat 840 cgccaatgca atcgactctc ttcctaagct gatctcccca tactttcatg tggaatactt 900 gccccccctg gatcacaact tccaaccacc cgtactccac aatcacgata cgcagagcga 960 ttcggtctcc tgtgcaatac gcaggatcac ccagagatgc aaaagggtat tcgtcacagg 1020 tgtcctaggc tcaactgagc tgttctggcc aaatgtaaat tcggcccctg acccatactg 1080 gcactcgttg gaatttctgg atatttggta ccatcccatt accccgggtg gcaagtggct 1140 atttggatta gacccatttc tccatttcgg gtcggcgcga atgcaggctc gcgatctcta 1200 ttcgcgtcct gagccagtag aggggcgtgc ggatgaagat caacaagcat gtcaatttcg 1260 ctatacagca atccagcaac tcatggatga gttctatgta gcggcagcac gagctgcggc 1320 aaatatgcca aagttgaaga ggttggcttt ggtggtaacc caccaaccga gttggagaat 1380 tgatggtgct cccctacacc atttcgagtt ccgggcgtcc aaagacaatc atgcctcaat 1440 tacttggacc tcctcaccag tatttactcc tagtcaggct gttatcgatg cctggatgcg 1500 tgtatcctgc attcgcggct tgtctatgcg agtaaggcta accggacgtc cattcaacga 1560 cagaggtaat acgcgctatg tggatcccac cgatgatgac cctgatagtg tgcatgaaga 1620 ggaggaggat gacgaggagt atgacgaggg ggatagcgag gaggatgaag gggaggaatg 1680 to 1681 <210> 49 <211> 1608 <212> DNA <213> Hypoxylon policicidum <400> 49 atggacaagc taccacagga aatcgtggac atcatcgtct tctttgccaa ctccaacgag 60 caagggaacc aatatcacat gcacggcccc gatgctaagc atcttgcgag acaattagtc 120 actgtttcca agaaattcca gcgcgccatc gagtgtgata cattcaaggt acttcatctc 180 gacttggacc gggctcgtga tgctcgtgat atcctttccc ggttctctag atggtcgcat 240 300. ctgagactac tagaatttac tgctatcctg cccgacttg acgaagttgc gtgcactcga 360. ttgagaacg ctgcagagaa agcagagaat tcgcgggtgt ttacggaaaa catcacagag ttgttccacc ttctctctca atgcccagtt gcatgtgctt ctagacctgt ggtattgagg 420 gtagatat attccaagac ggatcgcatc tgtgtcagcc acagtcttca aggccgtgtg 480 actcaaaagg agacgcacgc caagccagtg tctggtgaca taaggaggtg gaggtggaag 540 cggtcaaaac tacagcttga caagtcagtt cgtcttgcct caatcaaaca cctaaatata cctgtcttcc agatgcaaag gcgcccgtta ttattccgac atgttgctgc tgatagcatt 660 atgcgataa tatcagcaat gcctccctcc ctcaagttta ccgtcaaccg gttttcagat accgaaaaga acgatcttgc tcatcgaatt gcacatcgtg aggatcgc caatgcaatc gactctcttc ctaagctgat ctccccatac tttcatgtgg aatacttgcc ccccctggat 840 cacaacttcc aaccacccgt actccacaat cacgatacgc agagcgattc ggtctcctgt 900 gcaatacgca ggatcaccca gagatgcaaa agggtattcg tcacaggtgt cctaggctca actgagctgt tctggccaaa tgtaaattcg gcccctgacc catactggca ctcgttggaa 1020 tttctggata tttggtacca tcccattacc ccgggtggca agtggctatt tggattagac 1080 ccatttctcc attcgggtc ggcgcgaatg caggctcgcg atctctattc gcgtcctgag 1140 ccagtagagg ggcgtgcgga tgaagatcaa caagcatgtc aatttcgcta tacagcaatc 1200 cagcaactca tggatgagtt ctatgtagcg gcagcacgag ctgcggcaaa tatgccaaag 1260 ttgaagaggt tggctttggt ggtaacccac caaccgagtt ggagaattga tggtgctccc 1320 ctacaccatt tcgagttccg ggcgtccaaa gacaatcatg cctcaattac ttggacctcc 1380 tcaccagtat ttactcctag tcaggctgtt atcgatgcct ggatgcgtgt atcctgcatt 1440 cgcggcttgt ctatgcgagt aaggctaacc ggacgtccat tcaacgacag aggtaatacg 1500 cgctatgtgg atcccaccga tgatgaccct gatagtgtgc atgaagagga ggaggatgac 1560 gaggagtatg acgaggggga tagcgaggag gatgaagggg aggaatga 1608 <210> 50 <211> 535 <212> PRT <213> Hypoxylon policicidum <400> 50 Met Asp Lys Leu Pro Gln Glu Ile Val Asp Ile Ile Val Phe Phe Ala 1 5 10 15 Asn Ser Asn Glu Gln Gly Asn Gln Tyr His Met His Gly Pro Asp Ala 20 25 30 Lys His Leu Ala Arg Gln Leu Val Thr Val Ser Lys Lys Phe Gln Arg 35 40 45 Ala Ile Glu Cys Asp Thr Phe Lys Val Leu His Leu Asp Leu Asp Arg 50 55 60 Ala Arg Asp Ala Arg Asp Ile Leu Ser Arg Phe Ser Arg Trp Ser His 65 70 75 80 Leu Arg Leu Leu Glu Phe Thr Ala Ile Leu Pro Asp Tyr Asp Glu Val 85 90 95 Ala Cys Thr Arg Tyr Glu Asn Ala Ala Glu Lys Ala Glu Asn Ser Arg 100 105 110 Val Phe Thr Glu Asn Ile Thr Glu Leu Phe His Leu Leu Ser Gln Cys 115 120 125 Pro Val Ala Cys Ala Ser Arg Pro Val Val Leu Arg Val Glu Ile Tyr 130 135 140 Ser Lys Thr Asp Arg Ile Cys Val Ser His Ser Leu Gln Gly Arg Val 145 150 155 160 Thr Gln Lys Glu Thr His Ala Lys Pro Val Ser Gly Asp Ile Arg Arg 165 170 175 Trp Arg Trp Lys Arg Ser Lys Leu Gln Leu Asp Lys Ser Val Arg Leu 180 185 190 Ala Ser Ile Lys His Leu Asn Ile Pro Val Phe Gln Met Gln Arg Arg 195 200 205 Pro Leu Leu Phe Arg His Val Ala Ala Asp Ser Ile Met Arg Leu Ile 210 215 220 Ser Ala Met Pro Pro Ser Leu Lys Phe Thr Val Asn Arg Phe Ser Asp 225 230 235 240 Thr Glu Lys Asn Asp Leu Ala His Arg Ile Ala His Arg Glu Asp Ile 245 250 255 Ala Asn Ala Ile Asp Ser Leu Pro Lys Leu Ile Ser Pro Tyr Phe His 260 265 270 Val Glu Tyr Leu Pro Pro Leu Asp His Asn Phe Gln Pro Pro Val Leu 275 280 285 His Asn His Asp Thr Gln Ser Asp Ser Val Ser Cys Ala Ile Arg Arg 290 295 300 Ile Thr Gln Arg Cys Lys Arg Val Phe Val Thr Gly Val Leu Gly Ser 305 310 315 320 Thr Glu Leu Phe Trp Pro Asn Val Asn Ser Ala Pro Asp Pro Tyr Trp 325 330 335 His Ser Leu Glu Phe Leu Asp Ile Trp Tyr His Pro Ile Thr Pro Gly 340 345 350 Gly Lys Trp Leu Phe Gly Leu Asp Pro Phe Leu His Phe Gly Ser Ala 355 360 365 Arg Met Gln Ala Arg Asp Leu Tyr Ser Arg Pro Glu Pro Val Glu Gly 370 375 380 Arg Ala Asp Glu Asp Gln Gln Ala Cys Gln Phe Arg Tyr Thr Ala Ile 385 390 395 400 Gln Gln Leu Met Asp Glu Phe Tyr Val Ala Ala Ala Arg Ala Ala Ala 405 410 415 Asn Met Pro Lys Leu Lys Arg Leu Ala Leu Val Val Thr His Gln Pro 420 425 430 Ser Trp Arg Ile Asp Gly Ala Pro Leu His His Phe Glu Phe Arg Ala 435 440 445 Ser Lys Asp Asn His Ala Ser Ile Thr Trp Thr Ser Ser Pro Val Phe 450 455 460 Thr Pro Will Be Gln Ala Val Ile Asp Ala Trp Met Arg Will Be Cys Ile 465 470 475 480 Arg Gly Leu Ser Met Arg Val Arg Leu Thr Gly Arg Pro Phe Asn Asp 485,490,495 Arg Gly Asn Thr Arg Tyr Val Asp Pro Thr Asp Asp Pro Asp Ser 500 505 510 Val His Glu Glu Glu Glu Asp Asp Glu Glu Tyr Asp Glu Gly Asp Ser 515,520,525 Glu Glu Asp Glu Gly Glu Glu 530,535 <210> 51 <211> 819 <212> DNA <213> Penicillium paxilli <400> 51 tcaatttgct ttttcggcc cgcttatgcc aagtgactt tcgttacggt cgacgtacca 60 gaaacaatt ccataaaacc catcgattga taaaaacacc acagactcc acagtaccaa 120 aggactattc agccaaccga atgcttccga ccaatacatc caacgtaagc cggcaaaccc 180 aactgtacat gttgatccta agaagcggga agccctatag tacagagat gagtacttgt 240 atttctgtca ctttggggaa gaaattccga ttcatcgggc atgaatgag atcagacata 300 ccatagagta taggatgcgc cacgcgtact gcctcgacac aacagttgac tcaatccacc 360 aacgcttaga agaagttgac aaatgacggc ccccatgaa tatgccagcg caggcccgat 420 ttcaagggcc atgcacat gcccagagag aaagcccatg gtcgcgacga aaaatca 480 ggaaatattg cgctcaaca gcggcgcatg gccccactct cgggatgaa acgttattgc 540 tgcgtacatg acaccaagt tgatgaggag gcccatccag atacacccc tctccaccgg 600 gcttttcgag ggaaagacca gagagtagac cagttcccac gcgatgttgc aacagagggg 660 cataatcgac attccgtaag ttcatgttt gaacgagatg tagaccattc cgatgtagtt 720 tatgatccat cctactccca tgccacaac aaaagatca gccaatggct taatcgcctg 780 gtactccgga ggagcttggg aaacatcaa accgtccat 819 <210> 52 <211> 732 <212> DNA <213> Penicillium paxilli <400> 52 tcaatttgct ttttcggcc cgcttatgcc aagtgactt tcgttacggt cgacgtacca 60 gaaacaatt ccataaaacc catcgattga taaaaacacc acagactcc acagtaccaa 120 aggactattc agccaaccga atgcttccga ccaatacatc caacgtaagc cggcaaaccc 180 aactgtacat gttgatccta agaagcggga agcccataga gtataggatg cgccacgcgt 240 actgcctcga cacacagtt gactcaatcc accaacgctt agagaagtt gatagaatgac 300 ggccccccat gatatgcca gcgcaggccc gatttcagg gccaatgca catgcccaga 360 gagaaagccc atggtcgcga cgaaaaatat caagcgctca caagcggcgc 420 atggccccac tctcgggatg aaacgttat tgctgcgtac atgacaccaa agttgatgag 480 gaggcccatc cagaatacac ccctctccac cgggctttc gagggaaaga ccagacagta 540 gaccagttcc cacgcgatgt tgcacagag gggcataatc gataccgt aagtttcatg 600 tttgaacgag atgtagacca ttccgatgta gtttatgatc catcctactc ccatgccaac 660 aacaaaaaga tcagccaatg gcttaatcgc ctggtactcc ggaggagctt gggaaacatc 720 aaaaccgtcc at 732 <210> 53 <211> 243 <212> PRT <213> Penicillium paxilli <400> 53 Met Asp Gly Phe Asp Val Ser Gln Ala Pro Pro Glu Tyr Gln Ala Ile 1 5 10 15 Lys Pro Leu Ala Asp Leu Phe Val Val Gly Met Gly Val Gly Trp Ile 20 25 30 Ile Asn Tyr Ile Gly Met Val Tyr Ile Ser Phe Lys His Glu Thr Tyr 35 40 45 Gly Met Ser Ile Met Pro Leu Cys Cys Asn Ile Ala Trp Glu Leu Val 50 55 60 Tyr Cys Leu Val Phe Pro Ser Lys Ser Pro Val Glu Arg Gly Val Phe 65 70 75 80 Trp Met Gly Leu Leu Ile Asn Phe Gly Val Met Tyr Ala Ala Ile Thr 85 90 95 Phe Ser Ser Arg Glu Trp Gly His Ala Pro Leu Val Glu Arg Asn Ile 100 105 110 Ser Leu Ile Phe Phe Val Ala Thr Met Gly Phe Leu Ser Gly His Val 115 120 125 Ala Leu Ala Leu Glu Ile Gly Pro Ala Leu Ala Tyr Ser Trp Gly Ala 130 135 140 Val Ile Cys Gln Leu Leu Leu Ser Val Gly Gly Leu Ser Gln Leu Leu 145 150 155 160 Cys Arg Gly Served Thr Arg Gly Ala Served Tyr Thr Leu Trp Ala Served Arg 165 170 175 Phe Leu Gly Ser Thr Cys Thr Val Gly Phe Ala Gly Leu Arg Trp Met 180 185 190 Tyr Trp Ser Glu Ala Phe Gly Trp Leu Asn Ser Pro Leu Val Leu Trp 195 200 205 Ser Leu Val Val Phe Leu Ser Ile Asp Gly Phe Tyr Gly Ile Cys Phe 210 215 220 Trp Tyr Val Asp Arg Asn Glu Lys Ser Leu Gly Ile Ser Gly Pro Lys 225 230 235 240 Lys Only Asn <210> 54 <211> 5083 <212> DNA <213> Hypoxylon policicidum <400> 54 atgggatatg gaccaggtac agagccctct gcaggcgttt ctgattccgc attgaggggc 60 atgcaagaac tgacagcatt tttcacca gagcttcata agacattaac tcgcgtacta 120 tctagctcta gatctgatgg cacgtcatta ttcacggtc gttcattgat ctctgaatcg 180 actcccgtaa cctccgataa tgactcac ggcgttctg gcctatcac tctttacgaa 240 ccccatttc ctactagcgc tacggccgat atcgtcttcg tccatggatt aggcggaggt 300 agtcgaaaaa catggtccta ttcccccgac cggtaccact actggcctca agcatggctt 360 gcgaacgact cggacttcgc agatatgcgc atccactcct tcggctataa gtccgactgg 420 gctgagcgac agcaaagcat cctcaatatt cgcgacttcg ccgaatcgtt agtcggagag 480 ctgaagaaca atcccggcat acgacgtagt aatacacgca tcatattcgt ttgccatagt 540 atgggcggat gcgtcgccaa aaaggcctac attatatcac gccaggatcc gacctgcaag 600 gacttagctg acagagtcca ctcgatattc tttctaggca ctccacacag gggcagtgat 660 cttgcagtga tcctgaagag gttgtctgtc atcgcttggg gctccaagcc gttcgtctcc 720 gacctctgc ccgaatcgtc tacattgaga gatatcaacg acacatttcg tcactatgcg 780 tcggatctgc gcctctggtc cttttacgag acgattcccg ccaagcccag gattttgaac 840 aaaatcgtgg ttgagaggca ctcggcaacg cttggttacc caaacgaaga gattgttgct 900 atgaacgctg accaccgcca gttgtgcaaa ttcaaaagtc cggctgaccc taattacaag 960 atattacgaa atgccctcca cacggctgcc gatatgctaa ggtcattacc acctttggcc 1020 ttagccccct tagaacctcg tatcaacttg gcttccacac aacagcattc gaaccaagag 1080 aacgcaagcc aacgcttgag gtcctttcta ggcgtcgttg atacgccaga ggatgaactc 1140 catattcttc aactgtttca ggagccggga tcttgccggt ggttctcgga atcgaaagac 1200 ttcgtgtctt gggaatcgga agggtccccg agcatactgt ggctcacggg gagaccagcg 1260 tccggtaaat ccgtccttc tagccacgtc atagagcaag tgaagtctct aggtgccttt 1320 tgcagttact ttttcttcaa gcaaagggag gctagcaagt ctacgctgag cgattgtttc 1380 cgctccattg cctttcagat ggccatacaa gatagtgaag tgatgaataa gctacttcaa 1440 ctcgaaggcg aatggatgac ctgggatatg agcgacgagc tgagcgtctg gagaaggctt 1500 ttcgtcaatg ggatcttcaa gctgccatcc atctcacagc acttctggat aatcgacggt 1560 gtcgatgaat gttctaattt caactccctt tttactaaga gaatcctcgc tacgcttcct 1620 agaggtattc gccttttcgc cagcagccgc catctagaag aaatccggcg cggtttggca 1680 cctcttgagt cgcgtgtgaa cctgcagtcc gtgtctgaaa ccgacactct tggtgatatg 1740 cggattttcg tgacttcgaa gctcagaggg ctagatcgcc ttgaaaatga tgacagtatc 1800 aatacaatgt gcgagaaaat ccttgggaag gcatccggtt ctttcttatg ggtccgactc 1860 gtacttcaag agttcgaaaa tgcttggact agcgaagcca tggatgctgt cttggatgaa 1920 atacctgtag atcttcaaga catgtatcgc cggatgcttc tgttaatgga gaaggatacg 1980 cgtacaatca agctcgccaa atccatcctg acgtgggccg ttctggcatg ccgaccgctc 2040 accgttgacg agctacgtct tgctgtgaag ctcgacgtta atgagacgct gcagagtgta 2100 agcaaggcga ttcctagttt atgccaccaa ctagtctttg tcgacagcgc taatagggtg 2160 cacatcttac atgcaaccgc ccgggaattt cttctcgacg aaactcttca gtcagagttt 2220 gctcttcatg gtgttgagaa gcacgcctct cttggctctc tactgcttcg gtatctcacc 2280 aacaccgcct ttaagttccg tgcgccaccg cggcggcagg gctcaggatt tcgcggtttt 2340 gcgaagcccg ttcatagtgt ttctcctgat ctatccctat tggactacgc agcttgcttc 2400 ttctctgagc acatataccg aggcgcctcg agagatgata ccttgatggc cgaactgtgt 2460 gccttcctaa agggcagaac tatcctctca tggattgaac acatcgctag aaatggagat 2520 cttggaggta ttaccaggac ggcgacagac ttacgtggat atctcggccg gaaactggaa 2580 catcttcctc ccacagatcc ctcggcctac cttgttgact gctgggtggc tgatctaatc 2640 cgggttgctg ccaagtttcg atttcagttg ctgacctgtc catcgtctat tcattacatt 2700 atcccatcat tctgtccatc agacactatt atctcgagga cctttgccaa tggtactcga 2760 ccttcgacaa taacagtcaa gggtatccct caagccacgt gggacgactg cttaacccgt 2820 atcgattttg agcgaggtcg agctaccgcc ttgagctatg gtacccaatt cttcgctgtt 2880 ggtttgtcga ccgggcagat ttcgctcttc gatccgcact ctgtccagcc cgtggcgaac 2940 cttgtccatc ctgaaagggt caagatgctg gagtttagct ccgacggaga atatcttgcc 3000 tcctctggca ctagaacttt gcttgtttgg cagccgaaat gtgggatgca gaagttgtca 3060 ttcccattgc agtcggagct tatcgctttt gttttcttaa gcggtgacga atttctttgt 3120 gctttcgcat ctggtgagct aactaaatgg taagatcacc tattcagacc tcacgaatca 3180 caacgccgcc tttcaaagct tcgtctttag atacagtact gattcttgac aacttaggcg 3240 tcttgacacg ggcgaacatg aaacaatctc ctggagaagc atgtgtgata cggatattac 3300 cggtactctg gatattcctg agcagccgcc aagctgcgcg gcgttctcta cagggggtga 3360 gactgtgctg cttgctgtgg gttaccgaaa ataccccata ttcatctggg acgcattgga 3420 gctacaattg ctgggacaat gcggagctga cgaaaacaat gggatagatg acatgacatt 3480 taatccaaac cctgagatat ctgcgctagt ggtctcctac aatgacggca ggttatgcct 3540 cttcgattac acgaccatga tgccgacgtt tactcgacaa agcgtattcg cgaacagtat 3600 tgcctgttcc ccggacggtc gaagtctcgt taccggcagt gtcaggggta tcattgaggt 3660 attcaaattc gaccaggacc ataccgggaa cacggtgcta gtccctatat acaatatcca 3720 agctttagat gactcgatcc gcagcgtagc ctttagcgct gatggcctgc gcttcgttga 3780 ccttcacgac caacaatgcc gcatttgggc acccgtgtct ctgatgcgga aggataacga 3840 actggagagc gtaagtggca ttgcgccgct tcctgccaaa atagtcacca tggccgacga 3900 ctccaatatc acagaaatca cgagtgcctt agctgtgtcc tctaacggcg gccgcgtcat 3960 tgcaggtaag agtaacggcg aggtctccgc gttttcggct gtcgatggga aagaattagg 4020 agtgctctat tcgcatggac gttgcgtatc cgttgtcaac gtcacctag gagaagctcg 4080 taatttagtc atctcagcgg atgatgctgg cagggtgcta gtggtggaac tgacaacgtg 4140 cctgccagcc tcctcggccg cacagaagct gccagcggct catgttattc tagagcgccg 4200 attcggtgct gctggtgttg gtctactcgt taacccctcg gtggaccgcc ttctaattag 4260 tgggcgacat gtggatgaac tctgtgatct gccgtcgggc cgggttttgg gttcgatatc 4320 tcatgtcgtc ggtaccgcta catcgacaag agccgtagcc ttccagcatc cagccaacga 4380 ggcctggatc gtcttgatgc tcactaatac cgctcgggtt ttccgctggt ccgatttcca 4440 ggagttgact accgtgaacg gcatcctcct tcggaaacct tcgacagccg tagaggctcc 4500 tctccccacg cattcccggg ctttgaaaac accaaccacg ctcagctcat cgtatcattt 4560 taggcaagat tttgtcctcg agttgctcag aatacgccc tccgtagctc cacgactcta 4620 cgtttggccg gtgtcagcat ttgatccggc aacagcacag ccggatcacc gagcgcaggt 4680 tcctagggaa gtgaatcttg acgccatcaa tgccggaatc ttggctgtgc ttggaatcgt 4740 aggtcagtct acggttctcc ttatggacgc caatctttgg gtatacagca tggagctccg 4800 cccaacgcaa gccacaccac aggcccaccc tggctttccc cctgaaacga cgatagttca 4860 ggacggacct gagccaatca cgcaacaaat tctgccggtg tttacaagga gacacttttt 4920 cgccctgagc gagtggcgca cagccggcga agagttgagg tgcgcggtag cgcagcattc 4980 tggtggcccg agtttcattt ttgccagtag gcagtacatc gtcgtcgttc aaggcggttt 5040 ggagtttcc gaggacatgg ctgtcggcca gcagagtgcg tag 5083 <210> 55 <211> 4995 <212> DNA <213> Hypoxylon policicidum <400> 55 atgggatatg gaccaggtac aagagcctct gcaggcgttt ctgattccgc attgaggggc 60 atgcaagaac tgacagcatt ttttcacca gagcttcata agacattaac tcgcgtacta 120 tctagctcta gatctgatgg cacgtcatta tttcacggtc gttcattgat ctctgaatcg 180 actcccgtaa cctccgataa tgagactcac ggcgttcttg gcctatcaac tctttacgaa 240 ccccatttc ctactagcgc tacggccgat atcgtcttcg tccatggatt aggcggaggt 300 agtcgaaaaa catggtccta ttcccccgac cggtaccact actggcctca agcatggctt 360 gcgaacgact cggacttcgc agatatgcgc atccactcct tcggctataa gtccgactgg 420 gctgagcgac agcaaagcat cctcaatatt cgcgacttcg ccgaatcgtt agtcggagag 480 ctgaagaaca atcccggcat acgacgtagt aatacacgca tcatattcgt ttgccatagt 540 atgggcggat gcgtcgccaa aaaggcctac attatatcac gccaggatcc gacctgcaag 600 gacttagctg acagagtcca ctcgatattc tttctaggca ctccacacag gggcagtgat 660 cttgcagtga tcctgaagag gttgtctgtc atcgcttggg gctccaagcc gttcgtctcc 720 gacctctgc ccgaatcgtc tacattgaga gatatcaacg acacatttcg tcactatgcg 780 tcggatctgc gcctctggtc cttttacgag acgattcccg ccaagcccag gattttgaac 840 aaaatcgtgg ttgagaggca ctcggcaacg cttggttacc caaacgaaga gattgttgct 900 atgaacgctg accaccgcca gttgtgcaaa ttcaaaagtc cggctgaccc taattacaag 960 atattacgaa atgccctcca cacggctgcc gatatgctaa ggtcattacc acctttggcc 1020 ttagccccct tagaacctcg tatcaacttg gcttccacac aacagcattc gaaccaagag 1080 aacgcaagcc aacgcttgag gtcctttcta ggcgtcgttg atacgccaga ggatgaactc 1140 catattcttc aactgtttca ggagccggga tcttgccggt ggttctcgga atcgaaagac 1200 ttcgtgtctt gggaatcgga agggtccccg agcatactgt ggctcacggg gagaccagcg 1260 tccggtaaat ccgtccttc tagccacgtc atagagcaag tgaagtctct aggtgccttt 1320 tgcagttact ttttcttcaa gcaaagggag gctagcaagt ctacgctgag cgattgtttc 1380 cgctccattg cctttcagat ggccatacaa gatagtgaag tgatgaataa gctacttcaa 1440 ctcgaaggcg aatggatgac ctgggatatg agcgacgagc tgagcgtctg gagaaggctt 1500 ttcgtcaatg ggatcttcaa gctgccatcc atctcacagc acttctggat aatcgacggt 1560 gtcgatgaat gttctaattt caactccctt tttactaaga gaatcctcgc tacgcttcct 1620 agaggtattc gccttttcgc cagcagccgc catctagaag aaatccggcg cggtttggca 1680 cctcttgagt cgcgtgtgaa cctgcagtcc gtgtctgaaa ccgacactct tggtgatatg 1740 cggattttcg tgacttcgaa gctcagaggg ctagatcgcc ttgaaaatga tgacagtatc 1800 aatacaatgt gcgagaaaat ccttgggaag gcatccggtt ctttcttatg ggtccgactc 1860 gtacttcaag agttcgaaaa tgcttggact agcgaagcca tggatgctgt cttggatgaa 1920 atacctgtag atcttcaaga catgtatcgc cggatgcttc tgttaatgga gaaggatacg 1980 cgtacaatca agctcgccaa atccatcctg acgtgggccg ttctggcatg ccgaccgctc 2040 accgttgacg agctacgtct tgctgtgaag ctcgacgtta atgagacgct gcagagtgta 2100 agcaaggcga ttcctagttt atgccaccaa ctagtctttg tcgacagcgc taatagggtg 2160 cacatcttac atgcaaccgc ccgggaattt cttctcgacg aaactcttca gtcagagttt 2220 gctcttcatg gtgttgagaa gcacgcctct cttggctctc tactgcttcg gtatctcacc 2280 aacaccgcct ttaagttccg tgcgccaccg cggcggcagg gctcaggatt tcgcggtttt 2340 gcgaagcccg ttcatagtgt ttctcctgat ctatccctat tggactacgc agcttgcttc 2400 ttctctgagc acatataccg aggcgcctcg agagatgata ccttgatggc cgaactgtgt 2460 gccttcctaa agggcagaac tatcctctca tggattgaac acatcgctag aaatggagat 2520 cttggaggta ttaccaggac ggcgacagac ttacgtggat atctcggccg gaaactggaa 2580 catcttcctc ccacagatcc ctcggcctac cttgttgact gctgggtggc tgatctaatc 2640 cgggttgctg ccaagtttcg atttcagttg ctgacctgtc catcgtctat tcattacatt 2700 atcccatcat tctgtccatc agacactatt atctcgagga cctttgccaa tggtactcga 2760 ccttcgacaa taacagtcaa gggtatccct caagccacgt gggacgactg cttaacccgt 2820 atcgattttg agcgaggtcg agctaccgcc ttgagctatg gtacccaatt cttcgctgtt 2880 ggtttgtcga ccgggcagat ttcgctcttc gatccgcact ctgtccagcc cgtggcgaac 2940 cttgtccatc ctgaaagggt caagatgctg gagtttagct ccgacggaga atatcttgcc 3000 tcctctggca ctagaacttt gcttgtttgg cagccgaaat gtgggatgca gaagttgtca 3060 ttcccattgc agtcggagct tatcgctttt gttttcttaa gcggtgacga atttctttgt 3120 gctttcgcat ctggtgagct aactaaatgg cgtcttgaca cgggcgaaca tgaaacaatc 3180 tcctggagaa gcatgtgtga tacggatatt accggtactc tggatattcc tgagcagccg 3240 ccaagctgcg cggcgttctc tacagggggt gagactgtgc tgcttgctgt gggttaccga 3300 aaatacccca tattcatctg ggacgcattg gagctacaat tgctgggaca atgcggagct 3360 gacgaaaaca atgggataga tgacatgaca tttaatccaa accctgagat atctgcgcta 3420 gtggtctcct acaatgacgg caggttatgc ctcttcgatt acacgaccat gatgccgacg 3480 tttactcgac aaagcgtatt cgcgaacagt attgcctgtt ccccggacgg tcgaagtctc 3540 gttaccggca gtgtcagggg tatcattgag gtattcaaat tcgaccagga ccataccggg 3600 aacacggtgc tagtccctat atacaatatc caagctttag atgactcgat ccgcagcgta 3660 gcctttagcg ctgatggcct gcgcttcgtt gaccttcacg accaacaatg ccgcatttgg 3720 gcacccgtgt ctctgatgcg gaaggataac gaactggaga gcgtaagtgg cattgcgccg 3780 cttcctgcca aaatagtcac catggccgac gactccaata tcacagaaat cacgagtgcc 3840 ttagctgtgt cctctaacgg cggccgcgtc attgcaggta agagtaacgg cgaggtctcc 3900 gcgttttcgg ctgtcgatgg gaaagaatta ggagtgctct attcgcatgg acgttgcgta 3960 tccgttgtca acgtcaccct aggagaagct cgtaatttag tcatctcagc ggatgatgct 4020 ggcagggtgc tagtggtgga actgacaacg tgcctgccag cctcctcggc cgcacagaag 4080 ctgccagcgg ctcatgttat tctagagcgc cgattcggtg ctgctgttgt tggtctactc 4140 gttaacccct cggtggaccg ccttctaatt agtgggcgac atgtggatga actctgtgat 4200 ctgccgtcgg gccgggtttt gggttcgata tctcatgtcg tcggtaccgc tacatcgaca 4260 agagccgtag ccttccagca tccagccaac gaggcctgga tcgtcttgat gctcactaat 4320 accgctcggg ttttccgctg gtccgatttc caggagttga ctaccgtgaa cggcatcctc 4380 cttcggaaac cttcgacagc cgtagaggct cctctcccca cgcattcccg ggctttgaaa 4440 acaccaacca cgctcagctc atcgtatcat tttaggcaag attttgtcct cgagttgctc 4500 agaatatcgc cctccgtagc tccacgactc tacgtttggc cggtgtcagc atttgatccg 4560 gcaacagcac agccggatca ccgagcgcag gttcctaggg aagtgaatct tgacgccatc 4620 aatgccggaa tcttggctgt gcttggaatc gtaggtcagt ctacggttct ccttatggac 4680 gccaatcttt gggtatacag catggagctc cgcccaacgc aagccacacc acaggcccac 4740 cctggctttc cccctgaaac gacgatagtt caggacggac ctgagccaat cacgcaacaa 4800 attctgccgg tgtttacaag gagacacttt ttcgccctga gcgagtggcg cacagccggc 4860 gaagagttga ggtgcgcggt agcgcagcat tctggtggcc cgagtttcat ttttgccagt 4920 aggcagtaca tcgtcgtcgt tcaaggcggt ttggagtttt ccgaggacat ggctgtcggc 4980 cagcagagtg cgtag 4995 <210> 56 <211> 1664 <212> PRT <213> Hypoxylon pulicicidum <400> 56 Met Gly Tyr Gly Pro Gly Thr Arg Ala Ser Ala Gly Val Ser Asp Ser 1 5 10 15 Ala Leu Arg Gly Met Gln Glu Leu Thr Ala Phe Phe Ser Pro Glu Leu 20 25 30 His Lys Thr Leu Thr Arg Val Leu Ser Ser Ser Arg Ser Asp Gly Thr 35 40 45 Ser Leu Phe His Gly Arg Ser Leu Ile Ser Glu Ser Thr Pro Val Thr 50 55 60 Ser Asp Asn Glu Thr His Gly Val Leu Gly Leu Ser Thr Leu Tyr Glu 65 70 75 80 Pro Ser Phe Pro Thr Ser Ala Thr Ala Asp Ile Val Phe Val His Gly 85 90 95 Leu Gly Gly Gly Ser Arg Lys Thr Trp Ser Tyr Ser Pro Asp Arg Tyr 100 105 110 His Tyr Trp Pro Gln Ala Trp Leu Ala Asn Asp Ser Asp Phe Ala Asp 115 120 125 Met Arg Ile His Ser Phe Gly Tyr Lys Ser Asp Trp Ala Glu Arg Gln 130 135 140 Gln Ser Ile Leu Asn Ile Arg Asp Phe Ala Glu Ser Leu Val Gly Glu 145 150 155 160 Leu Lys Asn Asn Pro Gly Ile Arg Arg Ser Asn Thr Arg Ile Ile Phe 165 170 175 Val Cys His Ser Met Gly Gly Cys Val Ala Lys Lys Ala Tyr Ile Ile 180 185 190 Ser Arg Gln Asp Pro Thr Cys Lys Asp Leu Ala Asp Arg Val His Ser 195 200 205 Ile Phe Phe Leu Gly Thr Pro His Arg Gly Ser Asp Leu Ala Val Ile 210 215 220 Leu Lys Arg Leu Ser Val Ile Ala Trp Gly Ser Lys Pro Phe Val Ser 225 230 235 240 Asp Leu Leu Pro Glu Ser Ser Thr Leu Arg Asp Ile Asn Asp Thr Phe 245 250 255 Arg His Tyr Ala Ser Asp Leu Arg Leu Trp Ser Phe Tyr Glu Thr Ile 260 265 270 Pro Ala Lys Pro Arg Ile Leu Asn Lys Ile Val Val Glu Arg His Ser 275 280 285 Ala Thr Leu Gly Tyr Pro Asn Glu Glu Ile Val Ala Met Asn Ala Asp 290 295 300 His Arg Gln Leu Cys Lys Phe Lys Ser Pro Ala Asp Pro Asn Tyr Lys 305 310 315 320 Ile Leu Arg Asn Ala Leu His Thr Ala Ala Asp Met Leu Arg Ser Leu 325 330 335 Pro Pro Leu Ala Leu Ala Pro Leu Glu Pro Arg Ile Asn Leu Ala Ser 340 345 350 Thr Gln Gln His Ser Asn Gln Glu Asn Ala Ser Gln Arg Leu Arg Ser 355 360 365 Phe Leu Gly Val Val Asp Thr Pro Glu Asp Glu Leu His Ile Leu Gln 370 375 380 Leu Phe Gln Glu Pro Gly Ser Cys Arg Trp Phe Ser Glu Ser Lys Asp 385 390 395 400 Phe Val Ser Trp Glu Ser Glu Gly Ser Pro Ser Ile Leu Trp Leu Thr 405 410 415 Gly Arg Pro Ala Ser Gly Lys Ser Val Leu Ser Ser His Val Ile Glu 420 425 430 Gln Val Lys Ser Leu Gly Ala Phe Cys Ser Tyr Phe Phe Phe Lys Gln 435 440 445 Arg Glu Ala Ser Lys Ser Thr Leu Ser Asp Cys Phe Arg Ser Ile Ala 450 455 460 Phe Gln Met Ala Ile Gln Asp Ser Glu Val Met Asn Lys Leu Leu Gln 465 470 475 480 Leu Glu Gly Glu Trp Met Thr Trp Asp Met Ser Asp Glu Leu Ser Val 485 490 495 Trp Arg Arg Leu Phe Val Asn Gly Ile Phe Lys Leu Pro Ser Ile Ser 500 505 510 Gln His Phe Trp Ile Ile Asp Gly Val Asp Glu Cys Ser Asn Phe Asn 515 520 525 Ser Leu Phe Thr Lys Arg Ile Leu Ala Thr Leu Pro Arg Gly Ile Arg 530 535 540 Leu Phe Ala Ser Ser Arg His Leu Glu Glu Ile Arg Arg Gly Leu Ala 545 550 555 560 Pro Leu Glu Ser Arg Val Asn Leu Gln Ser Val Ser Glu Thr Asp Thr 565 570 575 Leu Gly Asp Met Arg Ile Phe Val Thr Ser Lys Leu Arg Gly Leu Asp 580 585 590 Arg Leu Glu Asn Asp Asp Ser Ile Asn Thr Met Cys Glu Lys Ile Leu 595 600 605 Gly Lys Ala Ser Gly Ser Phe Leu Trp Val Arg Leu Val Leu Gln Glu 610 615 620 Phe Glu Asn Ala Trp Thr Ser Glu Ala Met Asp Ala Val Leu Asp Glu 625 630 635 640 Ile Pro Val Asp Leu Gln Asp Met Tyr Arg Arg Met Leu Leu Leu Met 645 650 655 Glu Lys Asp Thr Arg Thr Ile Lys Leu Ala Lys Ser Ile Leu Thr Trp 660 665 670 Ala Val Leu Ala Cys Arg Pro Leu Thr Val Asp Glu Leu Arg Leu Ala 675 680 685 Val Lys Leu Asp Val Asn Glu Thr Leu Gln Ser Val Ser Lys Ala Ile 690 695 700 Pro Ser Leu Cys His Gln Leu Val Phe Val Asp Ser Ala Asn Arg Val 705 710 715 720 His Ile Leu His Ala Thr Ala Arg Glu Phe Leu Leu Asp Glu Thr Leu 725 730 735 Gln Ser Glu Phe Ala Leu His Gly Val Glu Lys His Ala Ser Leu Gly 740 745 750 Ser Leu Leu Leu Arg Tyr Leu Thr Asn Thr Ala Phe Lys Phe Arg Ala 755 760 765 Pro Pro Arg Arg Gln Gly Ser Gly Phe Arg Gly Phe Ala Lys Pro Val 770 775 780 His Ser Val Ser Pro Asp Leu Ser Leu Leu Asp Tyr Ala Ala Cys Phe 785 790 795 800 Phe Ser Glu His Ile Tyr Arg Gly Ala Ser Arg Asp Asp Thr Leu Met 805 810 815 Ala Glu Leu Cys Ala Phe Leu Lys Gly Arg Thr Ile Leu Ser Trp Ile 820 825 830 Glu His Ile Ala Arg Asn Gly Asp Leu Gly Gly Ile Thr Arg Thr Ala 835 840 845 Thr Asp Leu Arg Gly Tyr Leu Gly Arg Lys Leu Glu His Leu Pro Pro 850 855 860 Thr Asp Pro Ser Ala Tyr Leu Val Asp Cys Trp Val Ala Asp Leu Ile 865 870 875 880 Arg Val Ala Ala Lys Phe Arg Phe Gln Leu Leu Thr Cys Pro Ser Ser 885 890 895 Ile His Tyr Ile Ile Pro Ser Phe Cys Pro Ser Asp Thr Ile Ile Ser 900 905 910 Arg Thr Phe Ala Asn Gly Thr Arg Pro Ser Thr Ile Thr Val Lys Gly 915 920 925 Ile Pro Gln Ala Thr Trp Asp Asp Cys Leu Thr Arg Ile Asp Phe Glu 930 935 940 Arg Gly Arg Ala Thr Ala Leu Ser Tyr Gly Thr Gln Phe Phe Ala Val 945 950 955 960 Gly Leu Ser Thr Gly Gln Ile Ser Leu Phe Asp Pro His Ser Val Gln 965 970 975 Pro Val Ala Asn Leu Val His Pro Glu Arg Val Lys Met Leu Glu Phe 980 985 990 Ser Ser Asp Gly Glu Tyr Leu Ala Ser Ser Gly Thr Arg Thr Leu Leu 995 1000 1005 Val Trp Gln Pro Lys Cys Gly Met Gln Lys Leu Ser Phe Pro Leu 1010 1015 1020 Gln Ser Glu Leu Ile Ala Phe Val Phe Leu Ser Gly Asp Glu Phe 1025 1030 1035 Leu Cys Ala Phe Ala Ser Gly Glu Leu Thr Lys Trp Arg Leu Asp 1040 1045 1050 Thr Gly Glu His Glu Thr Ile Ser Trp Arg Ser Met Cys Asp Thr 1055 1060 1065 Asp Ile Thr Gly Thr Leu Asp Ile Pro Glu Gln Pro Pro Ser Cys 1070 1075 1080 Ala Ala Phe Ser Thr Gly Gly Glu Thr Val Leu Leu Ala Val Gly 1085 1090 1095 Tyr Arg Lys Tyr Pro Ile Phe Ile Trp Asp Ala Leu Glu Leu Gln 1100 1105 1110 Leu Leu Gly Gln Cys Gly Ala Asp Glu Asn Asn Gly Ile Asp Asp 1115 1120 1125 Met Thr Phe Asn Pro Asn Pro Glu Ile Ser Ala Leu Val Val Ser 1130 1135 1140 Tyr Asn Asp Gly Arg Leu Cys Leu Phe Asp Tyr Thr Thr Met Met 1145 1150 1155 Pro Thr Phe Thr Arg Gln Ser Val Phe Ala Asn Ser Ile Ala Cys 1160 1165 1170 Ser Pro Asp Gly Arg Ser Leu Val Thr Gly Ser Val Arg Gly Ile 1175 1180 1185 Ile Glu Val Phe Lys Phe Asp Gln Asp His Thr Gly Asn Thr Val 1190 1195 1200 Leu Val Pro Ile Tyr Asn Ile Gln Ala Leu Asp Asp Ser Ile Arg 1205 1210 1215 Ser Val Ala Phe Ser Ala Asp Gly Leu Arg Phe Val Asp Leu His 1220 1225 1230 Asp Gln Gln Cys Arg Ile Trp Ala Pro Val Ser Leu Met Arg Lys 1235 1240 1245 Asp Asn Glu Leu Glu Ser Val Ser Gly Ile Ala Pro Leu Pro Ala 1250 1255 1260 Lys Ile Val Thr Met Ala Asp Asp Ser Asn Ile Thr Glu Ile Thr 1265 1270 1275 Ser Ala Leu Ala Val Ser Ser Asn Gly Gly Arg Val Ile Ala Gly 1280 1285 1290 Lys Ser Asn Gly Glu Val Ser Ala Phe Ser Ala Val Asp Gly Lys 1295 1300 1305 Glu Leu Gly Val Leu Tyr Ser His Gly Arg Cys Val Ser Val Val 1310 1315 1320 Asn Val Thr Leu Gly Glu Ala Arg Asn Leu Val Ile Ser Ala Asp 1325 1330 1335 Asp Ala Gly Arg Val Leu Val Val Glu Leu Thr Thr Cys Leu Pro 1340 1345 1350 Ala Ser Ser Ala Ala Gln Lys Leu Pro Ala Ala His Val Ile Leu 1355 1360 1365 Glu Arg Arg Phe Gly Ala Ala Val Val Gly Leu Leu Val Asn Pro 1370 1375 1380 Ser Val Asp Arg Leu Leu Ile Ser Gly Arg His Val Asp Glu Leu 1385 1390 1395 Cys Asp Leu Pro Ser Gly Arg Val Leu Gly Ser Ile Ser His Val 1400 1405 1410 Val Gly Thr Ala Thr Ser Thr Arg Ala Val Ala Phe Gln His Pro 1415 1420 1425 Ala Asn Glu Ala Trp Ile Val Leu Met Leu Thr Asn Thr Ala Arg 1430 1435 1440 Val Phe Arg Trp Ser Asp Phe Gln Glu Leu Thr Thr Val Asn Gly 1445 1450 1455 Ile Leu Leu Arg Lys Pro Ser Thr Ala Val Glu Ala Pro Leu Pro 1460 1465 1470 Thr His Ser Arg Ala Leu Lys Thr Pro Thr Thr Leu Ser Ser Ser 1475 1480 1485 Tyr His Phe Arg Gln Asp Phe Val Leu Glu Leu Leu Arg Ile Ser 1490 1495 1500 Pro Ser Val Ala Pro Arg Leu Tyr Val Trp Pro Val Ser Ala Phe 1505 1510 1515 Asp Pro Ala Thr Ala Gln Pro Asp His Arg Ala Gln Val Pro Arg 1520 1525 1530 Glu Val Asn Leu Asp Ala Ile Asn Ala Gly Ile Leu Ala Val Leu 1535 1540 1545 Gly Ile Val Gly Gln Ser Thr Val Leu Leu Met Asp Ala Asn Leu 1550 1555 1560 Trp Val Tyr Ser Met Glu Leu Arg Pro Thr Gln Ala Thr Pro Gln 1565 1570 1575 Ala His Pro Gly Phe Pro Pro Glu Thr Thr Ile Val Gln Asp Gly 1580 1585 1590 Pro Glu Pro Ile Thr Gln Gln Ile Leu Pro Val Phe Thr Arg Arg 1595 1600 1605 His Phe Phe Ala Leu Ser Glu Trp Arg Thr Ala Gly Glu Glu Leu 1610 1615 1620 Arg Cys Ala Val Ala Gln His Ser Gly Gly Pro Ser Phe Ile Phe 1625 1630 1635 Ala Ser Arg Gln Tyr Ile Val Val Val Gln Gly Gly Leu Glu Phe 1640 1645 1650 Ser Glu Asp Met Ala Val Gly Gln Gln Ser Ala 1655 1660

Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and having P450 oxygenase activity.

2. An isolated polynucleotide encoding the polypeptide of claim 1.

3. An isolated polynucleotide having at least 70% nucleic acid sequence identity to SEQ ID NO: 2, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and having P450 oxygenase activity.

4. A transcription unit (TU) comprising the isolated polynucleotide of claim 3.

5. A vector encoding the isolated polypeptide of claim 1. A vector comprising the isolated polynucleotide of claim 2 or 3 or the TU of claim 4.

7. An isolated host cell comprising the isolated polypeptide of claim 1, the isolated polynucleotide of claim 2 or claim 3, the TU of claim 4 and / or the vector of claim 5 or claim 6.

8. A method for producing at least one polyarthrosporic acid, the method comprising heterologously expressing the isolated polypeptide of claim 1, the isolated polynucleotide of claim 2 or claim 3, the TU of claim 4 and / or the vector of claim 5 or claim 6 in an isolated host cell.

9. A method for producing at least one Hypoxylon spp. polypeptide, the method comprising heterologously expressing the isolated polynucleotide of claim 2 or claim 3 or the TU of claim 4 and / or the vector of claim 5 or claim 6 in an isolated host cell.

10. A method for producing at least one arthrosporic acid, the method comprising contacting a substrate comprising a carbohydrate with a recombinant cell transformed with a nucleic acid having at least 70% sequence identity to SEQ ID NO: 2, the nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, expression of the recombinant cell resulting in an increased level of P450 oxygenase activity compared to the cell prior to transformation, such that the substrate is metabolized to at least one arthrosporic acid.

11. An isolated Hypoxylon pulicicidum strain comprising at least one heterologous nucleic acid sequence encoding an enzyme in a biosynthetic pathway for producing arthrosporic acid A10, wherein the heterologous nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and having P450 oxygenase activity.

12. A method for producing polyarticular spore acid A10, the method comprising expressing at least one heterologous nucleic acid sequence in Hypoxylon pulicicidum, wherein the at least one heterologous nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 2 and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 in a biosynthetic pathway for producing NAA 10 and having P450 oxygenase activity.

13. The isolated polynucleotide of claim 2, which has at least 75% nucleic acid sequence identity to SEQ ID NO:

2.

14. The isolated polynucleotide of claim 2, which has at least 80% nucleic acid sequence identity to SEQ ID NO:

2.

15. The isolated polynucleotide of claim 2, which has at least 85% nucleic acid sequence identity to SEQ ID NO:

2.

16. The isolated polynucleotide of claim 2, which has at least 90% nucleic acid sequence identity to SEQ ID NO:

2.

17. The isolated polynucleotide of claim 2, which has at least 95% nucleic acid sequence identity to SEQ ID NO:

2.

18. The isolated polynucleotide of claim 2, which has at least 99% nucleic acid sequence identity to SEQ ID NO:2.

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