NOVEL VARIANT HYPOCREA JECORINA CBH1 CELLULASE

Abstract

Described herein are variants of H. jecorina CBH I, a Cel7 enzyme. The present invention provides novel cellobiohydrolases that have improved thermostability and reversibility.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 13/523,800, filed Jun. 14, 2013, now U.S. Pat. No. 8,679,817, which is a continuation of U.S. patent application Ser. No. 13/107,702, filed May 13, 2011, now U.S. Pat. No. 8,232,080, which is a division of U.S. patent application Ser. No. 10/641,678, filed Aug. 15, 2003, now U.S. Pat. No. 7,972,832, which claims priority to U.S. Provisional Application No. 60/404,063, filed Aug. 16, 2002 (Attorney Docket No. GC772P), to U.S. Provisional Application No. 60/458,853 filed Mar. 27, 2003 (Attorney Docket No. GC772-2P), to U.S. Provisional Application No. 60/456,368 filed Mar. 21, 2003 (Attorney Docket No. GC793P) and to U.S. Provisional Application No. 60/458,696 filed Mar. 27, 2003 (Attorney Docket No. GC793-2P).

FIELD OF THE INVENTION

[0003] The present invention relates to variant cellobiohydrolase enzymes and isolated nucleic acid sequences which encode polypeptides having cellobiohydrolase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing recombinant variant CBH polypeptides.

REFERENCES

[0004] 1. Sheehan and Himmel Biotechnology Progress 15, pp 817-827 (1999)

[0005] 2. Matti Linko Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases pp 9-11 (1993)

[0006] 3. Tuula T. Teeri Trends in Biotechnology 15, pp 160-167 (1997)

[0007] 4. T. T. Teeri et al. Spec. Publ.-R. Soc. Chem., 246 (Recent Advances in Carbohydrate Bioengineering), pp 302-308. (1999)

[0008] 5. PDB reference 2OVW: Sulzenbacher, G., Schulein, M., Davies, G. J. Biochemistry 36 pp. 5902 (1997)

[0009] PDB reference 1A39: Davies, G. J., Ducros, V., Lewis, R. J., Borchert, T. V., Schulein, M. Journal of Biotechnology 57 pp. 91 (1997)

[0010] 7. PDB reference 6CEL: Divne, C., Stahlberg, J., Teed, T. T., Jones, T. A. Journal of Molecular Biology 275 pp. 309 (1998)

[0011] 8. PDB reference 1EG1: Kleywegt, G. J., Zou, J. Y., Divne, C., Davies, G. J., Sinning, I., Stahlberg, J., Reinikainen, T., Srisodsuk, M., Teed, T. T., Jones, T. A. Journal of Molecular Biology 272 pp. 383 (1997)

[0012] 9. PDB reference 1DY4 (8CEL): J. Stahlberg, H. Henriksson, C. Divne, R. Isaksson, G. Pettersson, G. Johansson, T. A. Jones

BACKGROUND OF THE INVENTION

[0013] Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms, including bacteria, yeast and fungi, that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., J. Biol. Chem., vol. 276, no. 26, pp. 24309-24314, Jun. 29, 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., Bioresource Tech. 77:193-196, 2001). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., Biotechnol. Gen. Engineer. Rev. vol. 14, pp. 365-414, 1997).

[0014] Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC 3.2.1.21) ("BG"). (Knowles et al., TIBTECH 5, 255-261, 1987; Shulein, Methods Enzymol., 160, 25, pp. 234-243, 1988). Endoglucanases act mainly on the amorphous parts of the cellulose fibre, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, the presence of a cellobiohydrolase in a cellulase system is required for efficient solubilization of crystalline cellulose (Suurnakki, et al. Cellulose 7:189-209, 2000). Beta-glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides (Freer, J. Biol. Chem. vol. 268, no. 13, pp. 9337-9342, 1993).

[0015] Cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose, such that the cellulases are readily produced in large quantities via fermentation. Filamentous fungi play a special role since many yeast, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose. See, e.g., Aro et al., 2001; Aubert et al., 1988; Wood et al., Methods in Enzymology, vol. 160, no. 9, pp. 87-116, 1988, and Coughlan, et al., "Comparative Biochemistry of Fungal and Bacterial Cellulolytic Enzyme Systems" Biochemistry and Genetics of Cellulose Degradation, pp. 11-30 1988.

[0016] The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources including Trichoderma reesei which contains known genes for 2 CBHs, i.e., CBH I and CBH II, at least 8 EGs, i.e., EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII and EGVIII, and at least 5 BGs, i.e., BG1, BG2, BG3, BG4 and BG5.

[0017] In order to efficiently convert crystalline cellulose to glucose the complete cellulase system comprising components from each of the CBH, EG and BG classifications is required, with isolated components less effective in hydrolyzing crystalline cellulose (Filho et al., Can. J. Microbiol. 42:1-5, 1996). A synergistic relationship has been observed between cellulase components from different classifications. In particular, the EG-type cellulases and CBH-type cellulases synergistically interact to more efficiently degrade cellulose. See, e.g., Wood, Biochemical Society Transactions, 611^th Meeting, Galway, vol. 13, pp. 407-410, 1985.

[0018] Cellulases are known in the art to be useful in the treatment of textiles for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, for improving the feel and appearance of cotton fabrics, and the like (Kumar et al., Textile Chemist and Colorist, 29:37-42, 1997).

[0019] Cellulase-containing detergent compositions with improved cleaning performance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and 2,094,826) and for use in the treatment of fabric to improve the feel and appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757; GB App. No. 1,358,599; The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report, Vol. 24, pp. 54-61, 1986), have been described.

[0020] Hence, cellulases produced in fungi and bacteria have received significant attention. In particular, fermentation of Trichoderma spp. (e.g., Trichoderma longibrachiatum or Trichoderma reesei) has been shown to produce a complete cellulase system capable of degrading crystalline forms of cellulose.

[0021] Although cellulase compositions have been previously described, there remains a need for new and improved cellulase compositions for use in household detergents, stonewashing compositions or laundry detergents, etc. Cellulases that exhibit improved performance are of particular interest.

BRIEF SUMMARY OF THE INVENTION

[0022] The invention provides an isolated cellulase protein, identified herein as variant CBH I, and nucleic acids which encode a variant CBH I.

[0023] In one embodiment the invention is directed to a variant CBH I cellulase, wherein said variant comprises a substitution or deletion at a position corresponding to one or more of residues S8, Q17, G22, T41, N49, S57, N64, A68, A77, N89, S92, N103, A112, S113, E193, S196, M213, L225, T226, P227, T246, D249, R251, Y252, T255, D257, D259, S278, S279, K286, L288, E295, T296, S297, A299, N301, E325, T332, F338, S342, F352, T356, Y371, T380, Y381, V393, R394, S398, V403, S411, G430, G440, T445, T462, T484, Q487, and P491 in CBH I from Hypocrea jecorina (SEQ ID NO: 2). In first aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosyl hydrolase of family 7, and wherein said nucleic acid encodes a substitution at a residue which is sensitive to temperature stress in the polypeptide encoded by said nucleic acid, wherein said variant cellobiohydrolase is derived from H. jecorina cellobiohydrolase. In second aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosyl hydrolase of family 7, and wherein said nucleic acid encodes a substitution at a residue which is effects enzyme processitivity in the polypeptide encoded by said nucleic acid, wherein said variant cellobiohydrolase is derived from H. jecorina cellobiohydrolase. In third aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of a glycosyl hydrolase of family 7, and wherein said nucleic acid encodes a substitution at a residue which is effects product inhibition in the polypeptide encoded by said nucleic acid, wherein said variant cellobiohydrolase is derived from H. jecorina cellobiohydrolase.

[0024] In a second embodiment the invention is directed to a variant CBH I cellulose comprising a substitution at a position corresponding to one or more of residues S8P, Q17L, G22D, T411, N49S, S57N, N64D, A68T, A77D, N89D, S92T, N103I, A112E, S113(T/N/D), E193V, S196T, M213I, L225F, T226A, P227(L/T/A), T246(C/A), D249K, R251A, Y252(A/Q), T255P, D257E, D259W, S278P, S279N, K286M, L288F, E295K, T296P, S297T, A299E, N301(R/K), E325K, T332(K/Y/H), F338Y, S342Y, F352L, T356L, Y371C, T380G, Y381D, V393G, R394A, S398T, V403D, S411F, G430F, G440R, T462I, T484S, Q487L and/or P491L in CBH I from Hypocrea jecorina (SEQ ID NO: 2). In one aspect of this embodiment the variant CBH I cellulase further comprises a deletion at a position corresponding to T445 in CBH I from Hypocrea jecorina (SEQ ID NO: 2). In a second aspect of this embodiment the variant CBH I cellulase further comprises the deletion of residues corresponding to residues 382-393 in CBH I of Hypocrea jecorina (SEQ ID NO: 2).

[0025] In a third embodiment the invention is directed to a variant CBH I cellulase, wherein said variant comprises a substitution at a position corresponding to a residue selected from the group consisting of S8P, N49S, A68T, A77D, N89D, S92T, S113(N/D), L225F, P227(A/L/T), D249K, T255P, D257E, S279N, L288F, E295K, S297T, A299E, N301K, T332(K/Y/H), F338Y, T356L, V393G, G430F in CBH I from Hypocrea jecorina (SEQ ID NO: 2).

[0026] In a fourth embodiment the invention is directed to a variant CBH I consists essentially of the mutations selected from the group consisting of

[0027] i. A112E/T226A;

[0028] ii. S196T/S411F;

[0029] iii. E295K/S398T;

[0030] iv. T246C/Y371C;

[0031] v. T41I plus deletion at T445

[0032] vi. A68T/G440R/P491L;

[0033] vii. G22D/S278P/T296P;

[0034] viii. T246A/R251A/Y252A;

[0035] ix. T380G/Y381D/R394A;

[0036] x. T380G/Y381D/R394A plus deletion of 382-393, inclusive;

[0037] xi. Y252Q/D259W/S342Y;

[0038] xii. S113T/T255P/K286M;

[0039] xiii. P227L/E325K/Q487L;

[0040] xiv. P227T/T484S/F352L;

[0041] xv. Q17L/E193V/M2131/F352L;

[0042] xvi. S8P/N49S/A68T/S113N;

[0043] xvii. S8P/N49S/A68T/S113N/P227L;

[0044] xviii. T41I/A112E/P227L/S278P/T296P;

[0045] xix. S8P/N49S/A68T/A112E/T226A;

[0046] xx. S8P/N49S/A68T/A112E/P227L;

[0047] xxi. S8P/T41I/N49S/A68T/A112E/P227L;

[0048] xxii. G22D/N495/A68T/P227L/S278P/T296P;

[0049] xxiii. S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P;

[0050] xxiv. G22D/N49S/A68T/N103I/S113N/P227L/S278P/T296P;

[0051] xxv. G22D/N49S/A68T/N103I/A112E/P227L/S278P/T296P;

[0052] xxvi. G22D/N49S/N64D/A68T/N103I/S113N/S278P/T296P;

[0053] xxvii. S8P/G22D/T411/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P;

[0054] xxviii. S8P/G22D/T411/N49S/A68T/N103I/S113N/P227L/S278P/T296P/N301R;

[0055] xxix. S8P/G22D/T411/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P/N301R

[0056] xxx. S8P/G22D/T411/N49S/A68T/S113N/P227L/D249K/S278P/T296P/N30 1R;

[0057] xxxi. S8P/T411/N49S/S57N/A68T/S113N/P227L/D249K/S278P/T296P/N30 1R;

[0058] xxxii. S8P/G22 D/T411/N49S/A68T/S113N/P227L/D249K/S278P/N301R;

[0059] xxxiii. S8P/T411/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;

[0060] xxxiv. S8P/G22D/T411/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;

[0061] xxxv. S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F;

[0062] xxxvi. S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F;

[0063] xxxvii. S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/- T296P/N301R/E325K/S411F;

[0064] xxxviii. S8P/T411/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E- 325K/V403D/S411F/T462I;

[0065] xxxix. S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N3- 01R/E325K/V403D/S411F/T462I; in CBH I from Hypocrea jecorina (SEQ ID NO:2).

[0066] In an fifth embodiment the invention is directed to a vector comprising a nucleic acid encoding a variant CBH I. In another aspect there is a construct comprising the nucleic acid of encoding the variant CBH I operably linked to a regulatory sequence.

[0067] In a sixth embodiment the invention is directed to a host cell transformed with the vector comprising a nucleic acid encoding a CBH I variant.

[0068] In a seventh embodiment the invention is directed to a method of producing a CBH I variant comprising the steps of:

[0069] (a) culturing a host cell transformed with the vector comprising a nucleic acid encoding a CBH I variant in a suitable culture medium under suitable conditions to produce CBH I variant;

[0070] (b) obtaining said produced CBH I variant.

[0071] In an eighth embodiment the invention is directed to a detergent composition comprising a surfactant and a CBH I variant. In one aspect of this embodiment the detergent is a laundry detergent. In a second aspect of this embodiment the detergent is a dish detergent. In third aspect of this invention, the variant CBH I cellulase is used in the treatment of a cellulose containing textile, in particular, in the stonewashing or indigo dyed denim.

[0072] In a ninth embodiment the invention is directed to a feed additive comprising a CBH I variant.

[0073] In a tenth embodiment the invention is directed to a method of treating wood pulp comprising contacting said wood pulp with a CBH I variant.

[0074] In a eleventh embodiment the invention is directed to a method of converting biomass to sugars comprising contacting said biomass with a CBH I variant.

[0075] In an embodiment, the cellulase is derived from a fungus, bacteria or Actinomycete.

[0076] In another aspect, the cellulase is derived from a fungus. In a most preferred embodiment, the fungus is a filamentous fungus. It is preferred the filamentous fungus belong to Euascomycete, in particular, Aspergillus spp., Gliocladium spp., Fusarium spp., Acremonium spp., Myceliophtora spp., Verticillium spp., Myrothecium spp., or Penicillium spp. In a further aspect of this embodiment, the cellulase is a cellobiohydrolase.

[0077] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 is the nucleic acid (lower line; SEQ ID NO: 1) and amino acid (upper line; SEQ ID NO: 2) sequence of the wild type Cel7A (CBH I) from H. jecorina.

[0079] FIG. 2 is the 3-D structure of H. jecorina CBH I.

[0080] FIGS. 3A and 3B show the amino acid alignment of the Cel7 family members for which there were crystal structures available. The sequences are: 2OVW--Fusarium oxysporum Cel7B (SEQ ID NO:32), 1A39--Humicola insolens Cel7B (SEQ ID NO:33), 6CEL--Hypocrea jecorina Cel7A (SEQ ID NO:34), 1EG1--Hypocrea jecorina Cel7B (SEQ ID NO:35), and the consensus sequence (SEQ ID NO:77).

[0081] FIG. 4 illustrates the crystal structures from the catalytic domains of these four Cel7 homologues aligned and overlayed as described herein.

[0082] FIG. 5 A-M is the nucleic acid sequence and deduced amino acid sequence for eight single residue mutations and five multiple mutation variants (SEQ ID NOs:5-30).

[0083] FIG. 6 A-D is the nucleic acid sequence for pTrex2 (SEQ ID NO:31).

[0084] FIG. 7 A & B depicts the construction of the expression plasmid pTEX.

[0085] FIG. 8 A-K is the amino acid alignment of all 42 members of the Cel7 family (SEQ ID NOs:32-73), and the consensus sequence, which is SEQ ID NO:74.

[0086] FIG. 9A is a representation of the thermal profiles of the wild type and eight single residue variants. FIG. 9B is a representation of the thermal profiles of the wild type and five variants. Legend for FIG. 9B: Cel7A=wild-type H. jecorina CBH 1; N301K=N301K variant; 334=P227L variant; 340=S8P/N49S/A68T/S113N variant; 350=S8P/N49S/A68T/S113N/P227L variant; and 363=S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P variant.

[0087] FIG. 10 is the pRAX1 vector. This vector is based on the plasmid pGAPT2 except a 5259 bp HindIII fragment of Aspergillus nidulans genomic DNA fragment AMA1 sequence (Molecular Microbiology 1996 19:565-574) was inserted. Base 1 to 1134 contains Aspergillus niger glucoamylase gene promoter. Base 3098 to 3356 and 4950 to 4971 contains Aspergillus niger glucoamylase terminator. Aspergillus nidulans pyrG gene was inserted from 3357 to 4949 as a marker for fungal transformation. There is a multiple cloning site (MCS) into which genes may be inserted.

[0088] FIG. 11 is the pRAXdes2 vector backbone. This vector is based on the plasmid vector pRAX1. A Gateway cassette has been inserted into pRAX1 vector (indicated by the arrow on the interior of the circular plasmid). This cassette contains recombination sequence attR1 and attR2 and the selection marker catH and ccdB. The vector has been made according to the manual given in Gateway® Cloning Technology: version 1 page 34-38 and can only replicate in E. coli DB3.1 from Invitrogen; in other E. coli hosts the ccdB gene is lethal. First a PCR fragment is made with primers containing attB1/2 recombination sequences. This fragment is recombined with pDONR201 (commercially available from Invitrogen); this vector contains attP1/2 recombination sequences with catH and ccdB in between the recombination sites. The BP clonase enzymes from Invitrogen are used to recombine the PCR fragment in this so-called ENTRY vector, clones with the PCR fragment inserted can be selected at 50 μg/ml kanamycin because clones expressing ccdB do not survive. Now the att sequences are altered and called attL1 and attL2. The second step is to recombine this clone with the pRAXdes2 vector (containing attR1 and attR2 catH and ccdB in between the recombination sites). The LR clonase enzymes from Invitrogen are used to recombine the insert from the ENTRY vector in the destination vector. Only pRAXCBH1 vectors are selected using 100 μg/ml ampicillin because ccdB is lethal and the ENTRY vector is sensitive to ampicillin. By this method the expression vector is now prepared and can be used to transform A. niger.

[0089] FIG. 12 provides an illustration of the pRAXdes2cbh1 vector which was used for expression of the nucleic acids encoding the CBH1 variants in Aspergillus. A nucleic acid encoding a CBH1 enzyme homolog or variant was cloned into the vector by homologous recombination of the att sequences.

DETAILED DESCRIPTION

[0090] The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0091] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Practitioners are particularly directed to Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel F M et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0092] The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0093] All publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention.

I. DEFINITIONS

[0094] The term "polypeptide" as used herein refers to a compound made up of a single chain of amino acid residues linked by peptide bonds. The term "protein" as used herein may be synonymous with the term "polypeptide" or may refer, in addition, to a complex of two or more polypeptides.

[0095] "Variant" means a protein which is derived from a precursor protein (e.g., the native protein) by addition of one or more amino acids to either or both the C- and N-terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, or deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence. The preparation of an enzyme variant is preferably achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative enzyme. The variant CBH I enzyme of the invention includes peptides comprising altered amino acid sequences in comparison with a precursor enzyme amino acid sequence wherein the variant CBH enzyme retains the characteristic cellulolytic nature of the precursor enzyme but which may have altered properties in some specific aspect. For example, a variant CBH enzyme may have an increased pH optimum or increased temperature or oxidative stability but will retain its characteristic cellulolytic activity. It is contemplated that the variants according to the present invention may be derived from a DNA fragment encoding a cellulase variant CBH enzyme wherein the functional activity of the expressed cellulase derivative is retained. For example, a DNA fragment encoding a cellulase may further include a DNA sequence or portion thereof encoding a hinge or linker attached to the cellulase DNA sequence at either the 5' or 3' end wherein the functional activity of the encoded cellulase domain is retained.

[0096] "Equivalent residues" may also be defined by determining homology at the level of tertiary structure for a precursor cellulase whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of a cellulase and Hypocrea jecorina CBH(N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the cellulase in question to the H. jecorina CBH I. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

R factor = h Fo ( h ) - Fc ( h ) h Fo ( h ) ##EQU00001##

[0097] Equivalent residues which are functionally analogous to a specific residue of H. jecorina CBH I are defined as those amino acids of a cellulase which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the H. jecorina CBH I. Further, they are those residues of the cellulase (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of H. jecorina CBH. The crystal structure of H. jecorina CBH I is shown in FIG. 2.

[0098] The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein such as CBH I may be produced. The present invention contemplates every possible variant nucleotide sequence, encoding CBH I, all of which are possible given the degeneracy of the genetic code.

[0099] A "heterologous" nucleic acid construct or sequence has a portion of the sequence which is not native to the cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native cell.

[0100] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

[0101] Accordingly, an "expression cassette" or "expression vector" is a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0102] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes.

[0103] As used herein, the term "selectable marker-encoding nucleotide sequence" refers to a nucleotide sequence which is capable of expression in cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent, or under corresponding selective growth conditions.

[0104] As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter will generally be appropriate to the host cell in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") are necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0105] "Chimeric gene" or "heterologous nucleic acid construct", as defined herein refers to a non-native gene (i.e., one that has been introduced into a host) that may be composed of parts of different genes, including regulatory elements. A chimeric gene construct for transformation of a host cell is typically composed of a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in a selectable marker chimeric gene, to a selectable marker gene encoding a protein conferring antibiotic resistance to transformed cells. A typical chimeric gene of the present invention, for transformation into a host cell, includes a transcriptional regulatory region that is constitutive or inducible, a protein coding sequence, and a terminator sequence. A chimeric gene construct may also include a second DNA sequence encoding a signal peptide if secretion of the target protein is desired.

[0106] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors, linkers or primers for PCR are used in accordance with conventional practice.

[0107] As used herein, the term "gene" means the segment of DNA involved in producing a polypeptide chain, that may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0108] In general, nucleic acid molecules which encode the variant CBH I will hybridize, under moderate to high stringency conditions to the wild type sequence provided herein as SEQ ID NO:1. However, in some cases a CBH I-encoding nucleotide sequence is employed that possesses a substantially different codon usage, while the protein encoded by the CBH I-encoding nucleotide sequence has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence may be modified to facilitate faster expression of CBH I in a particular prokaryotic or eukaryotic expression system, in accordance with the frequency with which a particular codon is utilized by the host. Te'o, et al. (FEMS Microbiology Letters 190:13-19, 2000), for example, describes the optimization of genes for expression in filamentous fungi.

[0109] A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5° C. (5° below the Tm of the probe); "high stringency" at about 5-10° below the Tm; "moderate" or "intermediate stringency" at about 10-20° below the Tm of the probe; and "low stringency" at about 20-25° below the Tm. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify sequences having about 80% or more sequence identity with the probe.

[0110] Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, and in Ausubel, F. M., et al., 1993, expressly incorporated by reference herein). An example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.

[0111] As used herein, "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.

[0112] As used herein, the terms "transformed", "stably transformed" or "transgenic" with reference to a cell means the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

[0113] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[0114] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).

[0115] It follows that the term "CBH I expression" refers to transcription and translation of the cbh I gene, the products of which include precursor RNA, mRNA, polypeptide, post-translationally processed polypeptides, and derivatives thereof, including CBH I from related species such as Trichoderma koningii, Hypocrea jecorina (also known as Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride) and Hypocrea schweinitzii. By way of example, assays for CBH I expression include Western blot for CBH I protein, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assays for CBH I mRNA, and endoglucanase activity assays as described in Shoemaker S. P. and Brown R. D. Jr. (Biochim. Biophys. Acta, 1978, 523:133-146) and Schulein (Methods Enzymol., 160, 25, pp. 234-243, 1988).

[0116] The term "alternative splicing" refers to the process whereby multiple polypeptide isoforms are generated from a single gene, and involves the splicing together of nonconsecutive exons during the processing of some, but not all, transcripts of the gene. Thus a particular exon may be connected to any one of several alternative exons to form messenger RNAs. The alternatively-spliced mRNAs produce polypeptides ("splice variants") in which some parts are common while other parts are different.

[0117] The term "signal sequence" refers to a sequence of amino acids at the N-terminal portion of a protein which facilitates the secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.

[0118] By the term "host cell" is meant a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of the expression construct. Host cells for use in the present invention can be prokaryotic cells, such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells. In general, host cells are filamentous fungi.

[0119] The term "filamentous fungi" means any and all filamentous fungi recognized by those of skill in the art. A preferred fungus is selected from the group consisting of Aspergillus, Trichoderma, Fusarium, Chrysosporium, Penicillium, Humicola, Neurospora, or alternative sexual forms thereof such as Emericella, Hypocrea. It has now been demonstrated that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. See Kuhls et al., PNAS (1996) 93:7755-7760.

[0120] The term "cellooligosaccharide" refers to oligosaccharide groups containing from 2-8 glucose units and having β-1,4 linkages, e.g., cellobiose.

[0121] The term "cellulase" refers to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose and/or glucose. Numerous examples of cellulases, such as exoglucanases, exocellobiohydrolases, endoglucanases, and glucosidases have been obtained from cellulolytic organisms, particularly including fungi, plants and bacteria.

[0122] CBH I from Hypocrea jecorina is a member of the Glycosyl Hydrolase Family 7 (hence Cel 7) and, specifically, was the first member of that family identified in Hypocrea jecorina (hence Cel 7A). The Glycosyl Hydrolase Family 7 contains both Endoglucanases and Cellobiohydrolases/exoglucanases, and that CBH I is the latter. Thus, the phrases CBH I, CBH I-type protein and Cel 7 cellobiohydrolases may be used interchangeably herein.

[0123] The term "cellulose binding domain" as used herein refers to portion of the amino acid sequence of a cellulase or a region of the enzyme that is involved in the cellulose binding activity of a cellulase or derivative thereof. Cellulose binding domains generally function by non-covalently binding the cellulase to cellulose, a cellulose derivative or other polysaccharide equivalent thereof. Cellulose binding domains permit or facilitate hydrolysis of cellulose fibers by the structurally distinct catalytic core region, and typically function independent of the catalytic core. Thus, a cellulose binding domain will not possess the significant hydrolytic activity attributable to a catalytic core. In other words, a cellulose binding domain is a structural element of the cellulase enzyme protein tertiary structure that is distinct from the structural element which possesses catalytic activity. Cellulose binding domain and cellulose binding module may be used interchangeably herein.

[0124] As used herein, the term "surfactant" refers to any compound generally recognized in the art as having surface active qualities. Thus, for example, surfactants comprise anionic, cationic and nonionic surfactants such as those commonly found in detergents. Anionic surfactants include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholytic surfactants include quaternary ammonium salt sulfonates, and betaine-type ampholytic surfactants. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule. Nonionic surfactants may comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.

[0125] As used herein, the term "cellulose containing fabric" refers to any sewn or unsewn fabrics, yarns or fibers made of cotton or non-cotton containing cellulose or cotton or non-cotton containing cellulose blends including natural cellulosics and manmade cellulosics (such as jute, flax, ramie, rayon, and lyocell).

[0126] As used herein, the term "cotton-containing fabric" refers to sewn or unsewn fabrics, yarns or fibers made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, raw cotton and the like.

[0127] As used herein, the term "stonewashing composition" refers to a formulation for use in stonewashing cellulose containing fabrics. Stonewashing compositions are used to modify cellulose containing fabrics prior to sale, i.e., during the manufacturing process. In contrast, detergent compositions are intended for the cleaning of soiled garments and are not used during the manufacturing process.

[0128] As used herein, the term "detergent composition" refers to a mixture which is intended for use in a wash medium for the laundering of soiled cellulose containing fabrics. In the context of the present invention, such compositions may include, in addition to cellulases and surfactants, additional hydrolytic enzymes, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, cellulase activators, antioxidants, and solubilizers.

[0129] As used herein, the term "decrease or elimination in expression of the cbh1 gene" means that either that the cbh1 gene has been deleted from the genome and therefore cannot be expressed by the recombinant host microorganism; or that the cbh1 gene has been modified such that a functional CBH1 enzyme is not produced by the host microorganism.

[0130] The term "variant cbh1 gene" or "variant CBH1" means, respectively, that the nucleic acid sequence of the cbh1 gene from H. jecorina has been altered by removing, adding, and/or manipulating the coding sequence or the amino acid sequence of the expressed protein has been modified consistent with the invention described herein.

[0131] As used herein, the term "purifying" generally refers to subjecting transgenic nucleic acid or protein containing cells to biochemical purification and/or column chromatography.

[0132] As used herein, the terms "active" and "biologically active" refer to a biological activity associated with a particular protein and are used interchangeably herein. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art.

[0133] As used herein, the term "enriched" means that the CBH is found in a concentration that is greater relative to the CBH concentration found in a wild-type, or naturally occurring, fungal cellulase composition. The terms enriched, elevated and enhanced may be used interchangeably herein.

[0134] A wild type fungal cellulase composition is one produced by a naturally occurring fungal source and which comprises one or more BGL, CBH and EG components wherein each of these components is found at the ratio produced by the fungal source. Thus, an enriched CBH composition would have CBH at an altered ratio wherein the ratio of CBH to other cellulase components (i.e., EGs, beta-glucosidases and other endoglucanases) is elevated. This ratio may be increased by either increasing CBH or decreasing (or eliminating) at least one other component by any means known in the art.

[0135] Thus, to illustrate, a naturally occurring cellulase system may be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient. The purified CBH may then be added to the enzymatic solution resulting in an enriched CBH solution. It is also possible to elevate the amount of CBH I produced by a microbe using molecular genetics methods to overexpress the gene encoding CBH, possibly in conjunction with deletion of one or more genes encoding other cellulases.

[0136] Fungal cellulases may contain more than one CBH component. The different components generally have different isoelectric points which allow for their separation via ion exchange chromatography and the like. Either a single CBH component or a combination of CBH components may be employed in an enzymatic solution.

[0137] When employed in enzymatic solutions, the homolog or variant CBH1 component is generally added in an amount sufficient to allow the highest rate of release of soluble sugars from the biomass. The amount of homolog or variant CBH1 component added depends upon the type of biomass to be saccharified which can be readily determined by the skilled artisan. However, when employed, the weight percent of the homolog or variant CBH1 component relative to any EG type components present in the cellulase composition is from preferably about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 30 weight percent, from about 15 to about 45 weight percent, from about 15 to about 50 weight percent.

II. HOST ORGANISMS

[0138] Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota. The filamentous fungi are characterized by vegetative mycelium having a cell wall composed of chitin, glucan, chitosan, mannan, and other complex polysaccharides, with vegetative growth by hyphal elongation and carbon catabolism that is obligately aerobic.

[0139] In the present invention, the filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, e.g., Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicola insolens and Humicola grisea; Chrysosporium sp., including C. lucknowense; Gliocladium sp.; Aspergillus sp.; Fusarium sp., Neurospora sp., Hypocrea sp., and Emericella sp. As used herein, the term "Trichoderma" or "Trichoderma sp." refers to any fungal strains which have previously been classified as Trichoderma or are currently classified as Trichoderma.

[0140] In one preferred embodiment, the filamentous fungal parent cell is an Aspergillus niger, Aspergillus awamori, Aspergillus aculeatus, or Aspergillus nidulans cell.

[0141] In another preferred embodiment, the filamentous fungal parent cell is a Trichoderma reesei cell.

III. CELLULASES

[0142] Cellulases are known in the art as enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As set forth above, cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases (EC 3.2.1.21) ("BG"). (Knowles, et al., TIBTECH 5, 255-261, 1987; Schulein, 1988).

[0143] Certain fungi produce complete cellulase systems which include exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases and beta-glucosidases or BG-type cellulases (Schulein, 1988). However, sometimes these systems lack CBH-type cellulases and bacterial cellulases also typically include little or no CBH-type cellulases. In addition, it has been shown that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. See, e.g., Wood, 1985. The different components, i.e., the various endoglucanases and exocellobiohydrolases in a multi-component or complete cellulase system, generally have different properties, such as isoelectric point, molecular weight, degree of glycosylation, substrate specificity and enzymatic action patterns.

[0144] It is believed that endoglucanase-type cellulases hydrolyze internal beta-1,4-glucosidic bonds in regions of low crystallinity of the cellulose and exo-cellobiohydrolase-type cellulases hydrolyze cellobiose from the reducing or non-reducing end of cellulose. It follows that the action of endoglucanase components can greatly facilitate the action of exo-cellobiohydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components. Further, beta-glucosidase-type cellulases have been shown to catalyze the hydrolysis of alkyl and/or aryl β-D-glucosides such as methyl β-D-glucoside and p-nitrophenyl glucoside as well as glycosides containing only carbohydrate residues, such as cellobiose. This yields glucose as the sole product for the microorganism and reduces or eliminates cellobiose which inhibits cellobiohydrolases and endoglucanases.

[0145] Cellulases also find a number of uses in detergent compositions including to enhance cleaning ability, as a softening agent and to improve the feel of cotton fabrics (Hemmpel, ITB Dyeing/Printing/Finishing 3:5-14, 1991; Tyndall, Textile Chemist and Colorist 24:23-26, 1992; Kumar et al., Textile Chemist and Colorist, 29:37-42, 1997). While the mechanism is not part of the invention, softening and color restoration properties of cellulase have been attributed to the alkaline endoglucanase components in cellulase compositions, as exemplified by U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757, which disclose that detergent compositions containing a cellulase composition enriched in a specified alkaline endoglucanase component impart color restoration and improved softening to treated garments as compared to cellulase compositions not enriched in such a component. In addition, the use of such alkaline endoglucanase components in detergent compositions has been shown to complement the pH requirements of the detergent composition (e.g., by exhibiting maximal activity at an alkaline pH of 7.5 to 10, as described in U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757).

[0146] Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced strength loss in the fabric (U.S. Pat. No. 4,822,516), contributing to reluctance to use cellulase compositions in commercial detergent applications. Cellulase compositions comprising endoglucanase components have been suggested to exhibit reduced strength loss for cotton-containing fabrics as compared to compositions comprising a complete cellulase system.

[0147] Cellulases have also been shown to be useful in degradation of cellulase biomass to ethanol (wherein the cellulase degrades cellulose to glucose and yeast or other microbes further ferment the glucose into ethanol), in the treatment of mechanical pulp (Pere et al., 1996), for use as a feed additive (WO 91/04673) and in grain wet milling.

[0148] Most CBHs and EGs have a multidomain structure consisting of a core domain separated from a cellulose binding domain (CBD) by a linker peptide (Suurnakki et al., 2000). The core domain contains the active site whereas the CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., 1986; Tomme et al., Eur. J. Biochem. 170:575-581, 1988). The CBDs are particularly important in the hydrolysis of crystalline cellulose. It has been shown that the ability of cellobiohydrolases to degrade crystalline cellulose clearly decreases when the CBD is absent (Linder and Teed, J. Biotechnol. 57:15-28, 1997). However, the exact role and action mechanism of CBDs is still a matter of speculation. It has been suggested that the CBD enhances the enzymatic activity merely by increasing the effective enzyme concentration at the surface of cellulose (Stahlberg et al., Bio/Technol. 9:286-290, 1991), and/or by loosening single cellulose chains from the cellulose surface (Tormo et al., EMBO J. vol. 15, no. 21, pp. 5739-5751, 1996). Most studies concerning the effects of cellulase domains on different substrates have been carried out with core proteins of cellobiohydrolases, as their core proteins can easily be produced by limited proteolysis with papain (Tomme et al., 1988). Numerous cellulases have been described in the scientific literature, examples of which include: from Trichoderma reesei: Shoemaker, S. et al., Bio/Technology, 1:691-696, 1983, which discloses CBHI; Teed, T. et al., Gene, 51:43-52, 1987, which discloses CBHII. Cellulases from species other than Trichoderma have also been described e.g., Ooi et al., Nucleic Acids Research, vol. 18, no. 19, 1990, which discloses the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus; Kawaguchi T et al., Gene 173(2):287-8, 1996, which discloses the cloning and sequencing of the cDNA encoding beta-glucosidase 1 from Aspergillus aculeatus; Sakamoto et al., Curr. Genet. 27:435-439, 1995, which discloses the cDNA sequence encoding the endoglucanase CMCase-1 from Aspergillus kawachii IFO 4308; Saarilahti et al., Gene 90:9-14, 1990, which discloses an endoglucanase from Erwinia carotovara; Spilliaert R, et al., Eur J. Biochem. 224(3):923-30, 1994, which discloses the cloning and sequencing of bglA, coding for a thermostable beta-glucanase from Rhodothermus marinu; and Halldorsdottir S et al., Appl Microbiol Biotechnol. 49(3):277-84, 1998, which discloses the cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. However, there remains a need for identification and characterization of novel cellulases, with improved properties, such as improved performance under conditions of thermal stress or in the presence of surfactants, increased specific activity, altered substrate cleavage pattern, and/or high level expression in vitro.

[0149] The development of new and improved cellulase compositions that comprise varying amounts CBH-type, EG-type and BG-type cellulases is of interest for use: (1) in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., "stone washing" or "biopolishing"); (2) in compositions for degrading wood pulp or other biomass into sugars (e.g., for bio-ethanol production); and/or (3) in feed compositions.

IV. MOLECULAR BIOLOGY

[0150] In one embodiment this invention provides for the expression of variant CBH I genes under control of a promoter functional in a filamentous fungus. Therefore, this invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994)).

[0151] A. Methods for Identifying Homologous CBH1 Genes

[0152] The nucleic acid sequence for the wild type H. jecorina CBH1 is shown in FIG. 1 (SEQ ID NO:1). The invention, in one aspect, encompasses a nucleic acid molecule encoding a CBH1 homolog described herein. The nucleic acid may be a DNA molecule.

[0153] Techniques that can be used to isolate CBH I encoding DNA sequences are well known in the art and include, but are not limited to, cDNA and/or genomic library screening with a homologous DNA probe and expression screening with activity assays or antibodies against CBH I. Any of these methods can be found in Sambrook, et al. or in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel, et al., ed. Greene Publishing and Wiley-Interscience, New York (1987) ("Ausubel").

[0154] B. Methods of Mutating CBH I Nucleic Acid Sequences

[0155] Any method known in the art that can introduce mutations is contemplated by the present invention.

[0156] The present invention relates to the expression, purification and/or isolation and use of variant CBH1. These enzymes are preferably prepared by recombinant methods utilizing the cbh gene from H. jecorina.

[0157] After the isolation and cloning of the cbh1 gene from H. jecorina, other methods known in the art, such as site directed mutagenesis, are used to make the substitutions, additions or deletions that correspond to substituted amino acids in the expressed CBH1 variant. Again, site directed mutagenesis and other methods of incorporating amino acid changes in expressed proteins at the DNA level can be found in Sambrook, et al. and Ausubel, et al.

[0158] DNA encoding an amino acid sequence variant of the H. jecorina CBH1 is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the H. jecorina CBH1.

[0159] Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0160] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide, i.e., H. jecorina CBH1. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). See, also, for example Cadwell et al., PCR Methods and Applications, Vol 2, 28-33 (1992). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

[0161] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315-323 (1985). The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0162] Alternatively, or additionally, the desired amino acid sequence encoding a variant CBH I can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically.

[0163] The variant CBH I(s) so prepared may be subjected to further modifications, oftentimes depending on the intended use of the cellulase. Such modifications may involve further alteration of the amino acid sequence, fusion to heterologous polypeptide(s) and/or covalent modifications.

V. CBH1 NUCLEIC ACIDS AND CBH1 POLYPEPTIDES

[0164] A. Variant cbh-type Nucleic acids

[0165] The nucleic acid sequence for the wild type H. jecorina CBH I is shown in FIG. 1 (SEQ ID NO:1). The invention encompasses a nucleic acid molecule encoding the variant cellulases described herein. The nucleic acid may be a DNA molecule.

[0166] After the isolation and cloning of the CBH I, other methods known in the art, such as site directed mutagenesis, are used to make the substitutions, additions or deletions that correspond to substituted amino acids in the expressed CBH I variant. Again, site directed mutagenesis and other methods of incorporating amino acid changes in expressed proteins at the DNA level can be found in Sambrook, et al. and Ausubel, et al.

[0167] After DNA sequences that encode the CBH1 variants have been cloned into DNA constructs, the DNA is used to transform microorganisms. The microorganism to be transformed for the purpose of expressing a variant CBH1 according to the present invention may advantageously comprise a strain derived from Trichoderma sp. Thus, a preferred mode for preparing variant CBH1 cellulases according to the present invention comprises transforming a Trichoderma sp. host cell with a DNA construct comprising at least a fragment of DNA encoding a portion or all of the variant CBH1. The DNA construct will generally be functionally attached to a promoter. The transformed host cell is then grown under conditions so as to express the desired protein. Subsequently, the desired protein product is purified to substantial homogeneity.

[0168] However, it may in fact be that the best expression vehicle for a given DNA encoding a variant CBH1 may differ from H. jecorina. Thus, it may be that it will be most advantageous to express a protein in a transformation host that bears phylogenetic similarity to the source organism for the variant CBH1. In an alternative embodiment, Aspergillus niger can be used as an expression vehicle. For a description of transformation techniques with A. niger, see WO 98/31821, the disclosure of which is incorporated by reference in its entirety.

[0169] Accordingly, the present description of a Trichoderma spp. expression system is provided for illustrative purposes only and as one option for expressing the variant CBH1 of the invention. One of skill in the art, however, may be inclined to express the DNA encoding variant CBH1 in a different host cell if appropriate and it should be understood that the source of the variant CBH1 should be considered in determining the optimal expression host. Additionally, the skilled worker in the field will be capable of selecting the best expression system for a particular gene through routine techniques utilizing the tools available in the art.

[0170] B. Variant CBH1 Polypeptides

[0171] The amino acid sequence for the wild type H. jecorina CBH I is shown in FIG. 1 (SEQ ID NO:1). The variant CBH I polypeptides comprises a substitution or deletion at a position corresponding to one or more of residues S8, Q17, G22, T41, N49, S57, N64, A68, A77, N89, S92, N103, A112, S113, E193, S196, M213, L225, T226, P227, T246, D249, R251, Y252, T255, D257, D259, S278, S279, K286, L288, E295, T296, S297, A299, N301, E325, T332, F338, S342, F352, T356, Y371, T380, Y381, V393, R394, S398, V403, S411, G430, G440, T445, T462, T484, Q487, and P491 in CBH I from Hypocrea jecorina. Furthermore, the variant may further comprises a deletion of residues corresponding to residues 382-393 in CBH I from Hypocrea jecorina.

[0172] The variant CBH I's of this invention have amino acid sequences that are derived from the amino acid sequence of a precursor CBH I. The amino acid sequence of the CBH I variant differs from the precursor CBH I amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. In a preferred embodiment, the precursor CBH I is Hypocrea jecorina CBH I. The mature amino acid sequence of H. jecorina CBH I is shown in FIG. 1. Thus, this invention is directed to CBH I variants which contain amino acid residues at positions which are equivalent to the particular identified residue in H. jecorina CBH I. A residue (amino acid) of an CBH I homolog is equivalent to a residue of Hypocrea jecorina CBH I if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or is functionally analogous to a specific residue or portion of that residue in Hypocrea jecorina CBH I (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally). As used herein, numbering is intended to correspond to that of the mature CBH I amino acid sequence as illustrated in FIG. 1. In addition to locations within the precursor CBH I, specific residues in the precursor CBH I corresponding to the amino acid positions that are responsible for instability when the precursor CBH I is under thermal stress are identified herein for substitution or deletion. The amino acid position number (e.g., +51) refers to the number assigned to the mature Hypocrea jecorina CBH I sequence presented in FIG. 1.

[0173] The variant CBH1's of this invention have amino acid sequences that are derived from the amino acid sequence of a precursor H. jecorina CBH1. The amino acid sequence of the CBH1 variant differs from the precursor CBH1 amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. The mature amino acid sequence of H. jecorina CBH1 is shown in FIG. 1. Thus, this invention is directed to CBH1 variants which contain amino acid residues at positions which are equivalent to the particular identified residue in H. jecorina CBH1. A residue (amino acid) of an CBH1 variant is equivalent to a residue of Hypocrea jecorina CBH1 if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or is functionally analogous to a specific residue or portion of that residue in Hypocrea jecorina CBH1 (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally). As used herein, numbering is intended to correspond to that of the mature CBH1 amino acid sequence as illustrated in FIG. 1. In addition to locations within the precursor CBH1, specific residues in the precursor CBH1 corresponding to the amino acid positions that are responsible for instability when the precursor CBH1 is under thermal stress are identified herein for substitution or deletion. The amino acid position number (e.g., +51) refers to the number assigned to the mature Hypocrea jecorina CBH1 sequence presented in FIG. 1.

[0174] Alignment of amino acid sequences to determine homology is preferably determined by using a "sequence comparison algorithm." Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), by visual inspection or MOE by Chemical Computing Group, Montreal Canada.

[0175] An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

[0176] The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0177] Additional specific strategies for modifying stability of CBH1 cellulases are provided below:

[0178] (1) Decreasing the entropy of main-chain unfolding may introduce stability to the enzyme. For example, the introduction of proline residues may significantly stabilize the protein by decreasing the entropy of the unfolding (see, e.g., Watanabe, et al., Eur. J. Biochem. 226:277-283 (1994)). Similarly, glycine residues have no β-carbon, and thus have considerably greater backbone conformational freedom than many other residues. Replacement of glycines, preferably with alanines, may reduce the entropy of unfolding and improve stability (see, e.g., Matthews, et al., Proc. Natl. Acad. Sci. USA 84; 6663-6667 (1987)). Additionally, by shortening external loops it may be possible to improve stability. It has been observed that hyperthermophile produced proteins have shorter external loops than their mesophilic homologues (see, e.g., Russel, et al., Current Opinions in Biotechnology 6:370-374 (1995)). The introduction of disulfide bonds may also be effective to stabilize distinct tertiary structures in relation to each other. Thus, the introduction of cysteines at residues accessible to existing cysteines or the introduction of pairs of cysteines that could form disulfide bonds would alter the stability of a CBH1 variant.

[0179] (2) Decreasing internal cavities by increasing side-chain hydrophobicity may alter the stability of an enzyme. Reducing the number and volume of internal cavities increases the stability of enzyme by maximizing hydrophobic interactions and reducing packing defects (see, e.g., Matthews, Ann. Rev. Biochem. 62:139-160 (1993); Burley, et al., Science 229:23-29 (1985); Zuber, Biophys. Chem. 29:171-179 (1988); Kellis, et al., Nature 333:784-786 (1988)). It is known that multimeric proteins from thermophiles often have more hydrophobic sub-unit interfaces with greater surface complementarity than their mesophilic counterparts (Russel, et al., supra). This principle is believed to be applicable to domain interfaces of monomeric proteins. Specific substitutions that may improve stability by increasing hydrophobicity include lysine to arginine, serine to alanine and threonine to alanine (Russel, et al., supra). Modification by substitution to alanine or proline may increase side-chain size with resultant reduction in cavities, better packing and increased hydrophobicity. Substitutions to reduce the size of the cavity, increase hydrophobicity and improve the complementarity the interfaces between the domains of CBH1 may improve stability of the enzyme. Specifically, modification of the specific residue at these positions with a different residue selected from any of phenylalanine, tryptophan, tyrosine, leucine and isoleucine may improve performance.

[0180] (3) Balancing charge in rigid secondary structure, i.e., α-helices and β-turns may improve stability. For example, neutralizing partial positive charges on a helix N-terminus with negative charge on aspartic acid may improve stability of the structure (see, e.g., Eriksson, et al., Science 255:178-183 (1992)). Similarly, neutralizing partial negative charges on helix C-terminus with positive charge may improve stability. Removing positive charge from interacting with peptide N-terminus in β-turns should be effective in conferring tertiary structure stability. Substitution with a non-positively charged residue could remove an unfavorable positive charge from interacting with an amide nitrogen present in a turn.

[0181] (4) Introducing salt bridges and hydrogen bonds to stabilize tertiary structures may be effective. For example, ion pair interactions, e.g., between aspartic acid or glutamic acid and lysine, arginine or histidine, may introduce strong stabilizing effects and may be used to attach different tertiary structure elements with a resultant improvement in thermostability. Additionally, increases in the number of charged residue/non-charged residue hydrogen bonds, and the number of hydrogen-bonds generally, may improve thermostability (see, e.g., Tanner, et al., Biochemistry 35:2597-2609 (1996)). Substitution with aspartic acid, asparagine, glutamic acid or glutamine may introduce a hydrogen bond with a backbone amide. Substitution with arginine may improve a salt bridge and introduce an H-bond into a backbone carbonyl.

[0182] (5) Avoiding thermolabile residues in general may increase thermal stability. For example, asparagine and glutamine are susceptible to deamidation and cysteine is susceptible to oxidation at high temperatures. Reducing the number of these residues in sensitive positions may result in improved thermostability (Russel, et al., supra). Substitution or deletion by any residue other than glutamine or cysteine may increase stability by avoidance of a thermolabile residue.

[0183] (6) Stabilization or destabilization of binding of a ligand that confers modified stability to CBH1 variants. For example, a component of the matrix in which the CBH1 variants of this invention are used may bind to a specific surfactant/thermal sensitivity site of the CBH1 variant. By modifying the site through substitution, binding of the component to the variant may be strengthened or diminished. For example, a non-aromatic residue in the binding crevice of CBH1 may be substituted with phenylalanine or tyrosine to introduce aromatic side-chain stabilization where interaction of the cellulose substrate may interact favorably with the benzyl rings, increasing the stability of the CBH1 variant.

[0184] (7) Increasing the electronegativity of any of the surfactant/thermal sensitivity ligands may improve stability under surfactant or thermal stress. For example, substitution with phenylalanine or tyrosine may increase the electronegativity of D (aspartate) residues by improving shielding from solvent, thereby improving stability.

[0185] C. Anti-CBH Antibodies

[0186] The present invention further provides anti-CBH antibodies. The antibodies may be polyclonal, monoclonal, humanized, bispecific or heteroconjugate antibodies.

[0187] Methods of preparing polyclonal antibodies are known to the skilled artisan. The immunizing agent may be an CBH polypeptide or a fusion protein thereof. It may be useful to conjugate the antigen to a protein known to be immunogenic in the mammal being immunized. The immunization protocol may be determined by one skilled in the art based on standard protocols or routine experimentation.

[0188] Alternatively, the anti-CBH antibodies may be monoclonal antibodies. Monoclonal antibodies may be produced by cells immunized in an animal or using recombinant DNA methods. (See, e.g., Kohler et al., Nature, vol. 256, pp. 495-499, Aug. 7, 1975; U.S. Pat. No. 4,816,567).

[0189] An anti-CBH antibody of the invention may further comprise a humanized or human antibody. The term "humanized antibody" refers to humanized forms of non-human (e.g., murine) antibodies that are chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody. Methods for humanizing non-human antibodies are well known in the art, as further detailed in Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature, vol. 332, pp. 323-327, 1988; and Verhoeyen et al., Science, vol. 239, pp. 1534-1536, 1988. Methods for producing human antibodies are also known in the art. See, e.g., Jakobovits, A, et al., Annals New York Academy of Sciences, 764:525-535, 1995 and Jakobovits, A, Curr Opin Biotechnol 6(5):561-6, 1995.

VI. EXPRESSION OF RECOMBINANT CBH1 VARIANTS

[0190] The methods of the invention rely on the use cells to express variant CBH I, with no particular method of CBH I expression required.

[0191] The invention provides host cells which have been transduced, transformed or transfected with an expression vector comprising a variant CBH-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the parental host cell prior to transduction, transformation or transfection and will be apparent to those skilled in the art.

[0192] In one approach, a filamentous fungal cell or yeast cell is transfected with an expression vector having a promoter or biologically active promoter fragment or one or more (e.g., a series) of enhancers which functions in the host cell line, operably linked to a DNA segment encoding CBH, such that CBH is expressed in the cell line.

[0193] A. Nucleic Acid Constructs/Expression Vectors.

[0194] Natural or synthetic polynucleotide fragments encoding CBH I ("CBH I-encoding nucleic acid sequences") may be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into, and replication in, a filamentous fungal or yeast cell. The vectors and methods disclosed herein are suitable for use in host cells for the expression of CBH I. Any vector may be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Cloning and expression vectors are also described in Sambrook et al., 1989, Ausubel F M et al., 1989, and Strathern et al., The Molecular Biology of the Yeast Saccharomyces, 1981, each of which is expressly incorporated by reference herein. Appropriate expression vectors for fungi are described in van den Hondel, C.A.M.J. J. et al. (1991) In: Bennett, J. W. and Lasure, L. L. (eds.) More Gene Manipulations in Fungi. Academic Press, pp. 396-428. The appropriate DNA sequence may be inserted into a plasmid or vector (collectively referred to herein as "vectors") by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by standard procedures. Such procedures and related sub-cloning procedures are deemed to be within the scope of knowledge of those skilled in the art.

[0195] Recombinant filamentous fungi comprising the coding sequence for variant CBH I may be produced by introducing a heterologous nucleic acid construct comprising the variant CBH I coding sequence into the cells of a selected strain of the filamentous fungi.

[0196] Once the desired form of a variant cbh nucleic acid sequence is obtained, it may be modified in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence.

[0197] A selected variant cbh coding sequence may be inserted into a suitable vector according to well-known recombinant techniques and used to transform filamentous fungi capable of CBH I expression. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express variant CBH I. Therefore it is appreciated that such substitutions in the coding region fall within the sequence variants covered by the present invention. Any and all of these sequence variants can be utilized in the same way as described herein for a parent CBH I-encoding nucleic acid sequence.

[0198] The present invention also includes recombinant nucleic acid constructs comprising one or more of the variant CBH I-encoding nucleic acid sequences as described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.

[0199] Heterologous nucleic acid constructs may include the coding sequence for variant cbh: (i) in isolation; (ii) in combination with additional coding sequences; such as fusion protein or signal peptide coding sequences, where the cbh coding sequence is the dominant coding sequence; (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment in which the cbh coding sequence is a heterologous gene.

[0200] In one aspect of the present invention, a heterologous nucleic acid construct is employed to transfer a variant CBH I-encoding nucleic acid sequence into a cell in vitro, with established filamentous fungal and yeast lines preferred. For long-term, production of variant CBH I, stable expression is preferred. It follows that any method effective to generate stable transformants may be used in practicing the invention.

[0201] Appropriate vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as promoter and termination sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, i.e., promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified soluble protein antigen coding sequence is not normally expressed), operably linked to the coding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, many of which are commercially available and/or are described in Sambrook, et al., (supra).

[0202] Exemplary promoters include both constitutive promoters and inducible promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-1α promoter, a promoter containing the tet responsive element (TRE) in the tet-on or tet-off system as described (ClonTech and BASF), the beta actin promoter and the metallothionine promoter that can upregulated by addition of certain metal salts. A promoter sequence is a DNA sequence which is recognized by the particular filamentous fungus for expression purposes. It is operably linked to DNA sequence encoding a variant CBH I polypeptide. Such linkage comprises positioning of the promoter with respect to the initiation codon of the DNA sequence encoding the variant CBH I polypeptide in the disclosed expression vectors. The promoter sequence contains transcription and translation control sequence which mediate the expression of the variant CBH I polypeptide. Examples include the promoters from the Aspergillus niger, A awamori or A. oryzae glucoamylase, alpha-amylase, or alpha-glucosidase encoding genes; the A. nidulans gpdA or trpC Genes; the Neurospora crassa cbh1 or trp1 genes; the A. niger or Rhizomucor miehei aspartic proteinase encoding genes; the H. jecorina (T. reesei) cbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

[0203] The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art. Typical selectable marker genes include argB from A. nidulans or T. reesei, amdS from A. nidulans, pyr4 from Neurospora crassa or T. reesei, pyrG from Aspergillus niger or A. nidulans. Additional exemplary selectable markers include, but are not limited to trpc, trp1, oliC31, niaD or leu2, which are included in heterologous nucleic acid constructs used to transform a mutant strain such as trp-, pyr-, leu- and the like.

[0204] Such selectable markers confer to transformants the ability to utilize a metabolite that is usually not metabolized by the filamentous fungi. For example, the amdS gene from H. jecorina which encodes the enzyme acetamidase that allows transformant cells to grow on acetamide as a nitrogen source. The selectable marker (e.g. pyrG) may restore the ability of an auxotrophic mutant strain to grow on a selective minimal medium or the selectable marker (e.g. olic31) may confer to transformants the ability to grow in the presence of an inhibitory drug or antibiotic.

[0205] The selectable marker coding sequence is cloned into any suitable plasmid using methods generally employed in the art. Exemplary plasmids include pUC18, pBR322, pRAX and pUC100. The pRAX plasmid contains AMA1 sequences from A. nidulans, which make it possible to replicate in A. niger.

[0206] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., 1989; Freshney, Animal Cell Culture, 1987; Ausubel, et al., 1993; and Coligan et al., Current Protocols in Immunology, 1991.

[0207] B. Host Cells and Culture Conditions for CBH1 Production

Filamentous Fungi

[0208] Thus, the present invention provides filamentous fungi comprising cells which have been modified, selected and cultured in a manner effective to result in variant CBH I production or expression relative to the corresponding non-transformed parental fungi.

[0209] Examples of species of parental filamentous fungi that may be treated and/or modified for variant CBH I expression include, but are not limited to Trichoderma, e.g., Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii; Penicillium sp., Humicola sp., including Humicola insolens; Aspergillus sp., Chrysosporium sp., Fusarium sp., Hypocrea sp., and Emericella sp.

[0210] CBH I expressing cells are cultured under conditions typically employed to culture the parental fungal line. Generally, cells are cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., Appl. Environ. Microbiol. 63:1298-1306, 1997. Culture conditions are also standard, e.g., cultures are incubated at 28° C. in shaker cultures or fermenters until desired levels of CBH I expression are achieved.

[0211] Preferred culture conditions for a given filamentous fungus may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection (ATCC; atcc.org). After fungal growth has been established, the cells are exposed to conditions effective to cause or permit the expression of variant CBH I.

[0212] In cases where a CBH I coding sequence is under the control of an inducible promoter, the inducing agent, e.g., a sugar, metal salt or antibiotics, is added to the medium at a concentration effective to induce CBH I expression.

[0213] In one embodiment, the strain comprises Aspergillus niger, which is a useful strain for obtaining overexpressed protein. For example A. niger var awamori dgr246 is known to secrete elevated amounts of secreted cellulases (Goedegebuur et al, Curr. Genet (2002) 41: 89-98). Other strains of Aspergillus niger var awamori such as GCDAP3, GCDAP4 and GAP3-4 are known Ward et al (Ward, M, Wilson, L. J. and Kodama, K. H., 1993, Appl. Microbiol. Biotechnol. 39:738-743).

[0214] In another embodiment, the strain comprises Trichoderma reesei, which is a useful strain for obtaining overexpressed protein. For example, RL-P37, described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984) is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in overexpressing variant CBH1.

[0215] Where it is desired to obtain the variant CBH I in the absence of potentially detrimental native cellulolytic activity, it is useful to obtain a Trichoderma host cell strain which has had one or more cellulase genes deleted prior to introduction of a DNA construct or plasmid containing the DNA fragment encoding the variant CBH I. Such strains may be prepared by the method disclosed in U.S. Pat. No. 5,246,853 and WO 92/06209, which disclosures are hereby incorporated by reference. By expressing a variant CBH I cellulase in a host microorganism that is missing one or more cellulase genes, the identification and subsequent purification procedures are simplified. Any gene from Trichoderma sp. which has been cloned can be deleted, for example, the cbh1, cbh2, egl1, and egl2 genes as well as those encoding EG III and/or EGV protein (see e.g., U.S. Pat. No. 5,475,101 and WO 94/28117, respectively).

[0216] Gene deletion may be accomplished by inserting a form of the desired gene to be deleted or disrupted into a plasmid by methods known in the art. The deletion plasmid is then cut at an appropriate restriction enzyme site(s), internal to the desired gene coding region, and the gene coding sequence or part thereof replaced with a selectable marker. Flanking DNA sequences from the locus of the gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side of the selectable marker gene. An appropriate deletion plasmid will generally have unique restriction enzyme sites present therein to enable the fragment containing the deleted gene, including flanking DNA sequences, and the selectable marker gene to be removed as a single linear piece.

[0217] A selectable marker must be chosen so as to enable detection of the transformed microorganism. Any selectable marker gene that is expressed in the selected microorganism will be suitable. For example, with Aspergillus sp., the selectable marker is chosen so that the presence of the selectable marker in the transformants will not significantly affect the properties thereof. Such a selectable marker may be a gene that encodes an assayable product. For example, a functional copy of a Aspergillus sp. gene may be used which if lacking in the host strain results in the host strain displaying an auxotrophic phenotype. Similarly, selectable markers exist for Trichoderma sp.

[0218] In one embodiment, a pyrG.sup.- derivative strain of Aspergillus sp. is transformed with a functional pyrG gene, which thus provides a selectable marker for transformation. A pyrG.sup.- derivative strain may be obtained by selection of Aspergillus sp. strains that are resistant to fluoroorotic acid (FOA). The pyrG gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyrG gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyrG^derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges & Barreau, Curr. Genet. 19:359-365 (1991), and van Hartingsveldte et al., (1986) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol. Gen. Genet. 206:71-75). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyrG gene is preferably employed as a selectable marker.

[0219] In a second embodiment, a pyr4.sup.- derivative strain of Hyprocrea sp. (Hyprocrea sp. (Trichoderma sp.)) is transformed with a functional pyr4 gene, which thus provides a selectable marker for transformation. A pyr4.sup.- derivative strain may be obtained by selection of Hyprocrea sp. (Trichoderma sp.) strains that are resistant to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyr4.sup.- derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges & Barreau, Curr. Genet. 19:359-365 (1991)). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyr4 gene is preferably employed as a selectable marker.

[0220] To transform pyrG.sup.- Aspergillus sp. or pyr4.sup.- Hyprocrea sp. (Trichoderma sp.) so as to be lacking in the ability to express one or more cellulase genes, a single DNA fragment comprising a disrupted or deleted cellulase gene is then isolated from the deletion plasmid and used to transform an appropriate pyr Aspergillus or pyr Trichoderma host. Transformants are then identified and selected based on their ability to express the pyrG or pyr4, respectively, gene product and thus compliment the uridine auxotrophy of the host strain. Southern blot analysis is then carried out on the resultant transformants to identify and confirm a double crossover integration event that replaces part or all of the coding region of the genomic copy of the gene to be deleted with the appropriate pyr selectable markers.

[0221] Although the specific plasmid vectors described above relate to preparation of pyr.sup.- transformants, the present invention is not limited to these vectors. Various genes can be deleted and replaced in the Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) strain using the above techniques. In addition, any available selectable markers can be used, as discussed above. In fact, any host, e.g., Aspergillus sp. or Hyprocrea sp., gene that has been cloned, and thus identified, can be deleted from the genome using the above-described strategy.

[0222] As stated above, the host strains used may be derivatives of Hyprocrea sp. (Trichoderma sp.) that lack or have a nonfunctional gene or genes corresponding to the selectable marker chosen. For example, if the selectable marker of pyrG is chosen for Aspergillus sp., then a specific pyrG.sup.- derivative strain is used as a recipient in the transformation procedure. Also, for example, if the selectable marker of pyr4 is chosen for a Hyprocrea sp., then a specific pyr4.sup.- derivative strain is used as a recipient in the transformation procedure. Similarly, selectable markers comprising Hyprocrea sp. (Trichoderma sp.) genes equivalent to the Aspergillus nidulans genes amdS, argB, trpC, niaD may be used. The corresponding recipient strain must therefore be a derivative strain such as argB.sup.-, trpC.sup.-, niaD.sup.-, respectively.

[0223] DNA encoding the CBH I variant is then prepared for insertion into an appropriate microorganism. According to the present invention, DNA encoding a CBH I variant comprises the DNA necessary to encode for a protein that has functional cellulolytic activity. The DNA fragment encoding the CBH I variant may be functionally attached to a fungal promoter sequence, for example, the promoter of the glaA gene in Aspergillus or the promoter of the cbh1 or egl1 genes in Trichoderma.

[0224] It is also contemplated that more than one copy of DNA encoding a CBH I variant may be recombined into the strain to facilitate overexpression. The DNA encoding the CBH I variant may be prepared by the construction of an expression vector carrying the DNA encoding the variant. The expression vector carrying the inserted DNA fragment encoding the CBH I variant may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA of the host, typically a plasmid. In preferred embodiments two types of expression vectors for obtaining expression of genes are contemplated. The first contains DNA sequences in which the promoter, gene-coding region, and terminator sequence all originate from the gene to be expressed. Gene truncation may be obtained where desired by deleting undesired DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences. A selectable marker may also be contained on the vector allowing the selection for integration into the host of multiple copies of the novel gene sequences.

[0225] The second type of expression vector is preassembled and contains sequences required for high-level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general-purpose expression vector such that it is under the transcriptional control of the expression cassettes promoter and terminator sequences.

[0226] For example, in Aspergillus, pRAX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong glaA promoter.

[0227] For example, in Hypocrea, pTEX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong cbh1 promoter.

[0228] In the vector, the DNA sequence encoding the CBH I variant of the present invention should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence that shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. An optional signal peptide provides for extracellular production of the CBH I variant. The DNA encoding the signal sequence is preferably that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo-cellobiohydrolase or endoglucanase from Trichoderma, is contemplated in the present invention.

[0229] The procedures used to ligate the DNA sequences coding for the variant CBH I of the present invention with the promoter, and insertion into suitable vectors are well known in the art.

[0230] The DNA vector or construct described above may be introduced in the host cell in accordance with known techniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like.

[0231] In the preferred transformation technique, it must be taken into account that the permeability of the cell wall to DNA in Hyprocrea sp. (Trichoderma sp.) is very low. Accordingly, uptake of the desired DNA sequence, gene or gene fragment is at best minimal. There are a number of methods to increase the permeability of the Hyprocrea sp. (Trichoderma sp.) cell wall in the derivative strain (i.e., lacking a functional gene corresponding to the used selectable marker) prior to the transformation process.

[0232] The preferred method in the present invention to prepare Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) for transformation involves the preparation of protoplasts from fungal mycelium. See Campbell et al. Improved transformation efficiency of A. niger using homologous niaD gene for nitrate reductase. Curr. Genet. 16:53-56; 1989. The mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is preferable to use about a 1.2 M solution of sorbitol in the suspension medium.

[0233] Uptake of the DNA into the host strain, (Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.), is dependent upon the calcium ion concentration. Generally between about 10 mM CaCl₂ and 50 mM CaCl₂ is used in an uptake solution. Besides the need for the calcium ion in the uptake solution, other items generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membranes thus permitting the contents of the medium to be delivered into the cytoplasm of the host cell, by way of example either Aspergillus sp. or Hyprocrea sp. strain, and the plasmid DNA is transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA tenderly integrated into the host chromosome.

[0234] Usually a suspension containing the Aspergillus sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10⁵ to 10⁶/mL, preferably 2×10⁵/mL are used in transformation. Similarly, a suspension containing the Hyprocrea sp. (Trichoderma sp.) protoplasts or cells that have been subjected to a permeability treatment at a density of 10⁸ to 10⁹/mL, preferably 2×10⁸/mL are used in transformation. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl₂) are mixed with the desired DNA. Generally a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, it is preferable to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in transformation.

[0235] Generally, the mixture is then incubated at approximately 0° C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired gene or DNA sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is preferably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then incubated either at room temperature or on ice before the addition of a sorbitol and CaCl₂ solution. The protoplast suspension is then further added to molten aliquots of a growth medium. This growth medium permits the growth of transformants only. Any growth medium can be used in the present invention that is suitable to grow the desired transformants. However, if Pyr.sup.+ transformants are being selected it is preferable to use a growth medium that contains no uridine. The subsequent colonies are transferred and purified on a growth medium depleted of uridine.

[0236] At this stage, stable transformants may be distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium lacking uridine. Additionally, in some cases a further test of stability may made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium lacking uridine.

[0237] In a particular embodiment of the above method, the CBH I variant(s) are recovered in active form from the host cell after growth in liquid media either as a result of the appropriate post translational processing of the CBH I variant.

(ii) Yeast

[0238] The present invention also contemplates the use of yeast as a host cell for CBH I production. Several other genes encoding hydrolytic enzymes have been expressed in various strains of the yeast S. cerevisiae. These include sequences encoding for two endoglucanases (Penttila et al., Yeast vol. 3, pp 175-185, 1987), two cellobiohydrolases (Penttila et al., Gene, 63: 103-112, 1988) and one beta-glucosidase from Trichoderma reesei (Cummings and Fowler, Curr. Genet. 29:227-233, 1996), a xylanase from Aureobasidlium pullulans (Li and Ljungdahl, Appl. Environ. Microbiol. 62, no. 1, pp. 209-213, 1996), an alpha-amylase from wheat (Rothstein et al., Gene 55:353-356, 1987), etc. In addition, a cellulase gene cassette encoding the Butyrivibrio fibrisolvens endo-[beta]-1,4-glucanase (END1), Phanerochaete chrysosporium cellobiohydrolase (CBH1), the Ruminococcus flavefaciens cellodextrinase (CEL1) and the Endomyces fibrilizer cellobiase (Bgl1) was successfully expressed in a laboratory strain of S. cerevisiae (Van Rensburg et al., Yeast, vol. 14, pp. 67-76, 1998).

[0239] C. Introduction of an CBH I-Encoding Nucleic Acid Sequence into Host Cells

[0240] The invention further provides cells and cell compositions which have been genetically modified to comprise an exogenously provided variant CBH 1-encoding nucleic acid sequence. A parental cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc, as further described above.

[0241] The methods of transformation of the present invention may result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extrachromosomal transformation vector is also contemplated.

[0242] Many standard transfection methods can be used to produce Trichoderma reesei cell lines that express large quantities of the heterologus protein. Some of the published methods for the introduction of DNA constructs into cellulase-producing strains of Trichoderma include Lorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila and Kolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, for Streptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich, UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol. Lett. 55: 135-138).

[0243] Other methods for introducing a heterologous nucleic acid construct (expression vector) into filamentous fungi (e.g., H. jecorina) include, but are not limited to the use of a particle or gene gun, permeabilization of filamentous fungi cells walls prior to the transformation process (e.g., by use of high concentrations of alkali, e.g., 0.05 M to 0.4 M CaCl₂ or lithium acetate), protoplast fusion or agrobacterium mediated transformation. An exemplary method for transformation of filamentous fungi by treatment of protoplasts or spheroplasts with polyethylene glycol and CaCl₂ is described in Campbell, E. I. et al., Curr. Genet. 16:53-56, 1989 and Penttila, M. et al., Gene, 63:11-22, 1988.

[0244] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method described in U.S. Pat. No. 6,255,115. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the heterologous gene.

[0245] In addition, heterologous nucleic acid constructs comprising a variant CBH I-encoding nucleic acid sequence can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection.

[0246] The invention further includes novel and useful transformants of filamentous fungi such as H. jecorina and A. niger for use in producing fungal cellulase compositions. The invention includes transformants of filamentous fungi especially fungi comprising the variant CBH I coding sequence, or deletion of the endogenous cbh coding sequence.

[0247] Following introduction of a heterologous nucleic acid construct comprising the coding sequence for a variant cbh 1, the genetically modified cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying expression of a variant CBH I-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

[0248] The progeny of cells into which such heterologous nucleic acid constructs have been introduced are generally considered to comprise the variant CBH I-encoding nucleic acid sequence found in the heterologous nucleic acid construct.

[0249] The invention further includes novel and useful transformants of filamentous fungi such as H. jecorina for use in producing fungal cellulase compositions. The invention includes transformants of filamentous fungi especially fungi comprising the variant cbh 1 coding sequence, or deletion of the endogenous cbh coding sequence.

[0250] Stable transformants of filamentous fungi can generally be distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth rather than ragged outline on solid culture medium. Additionally, in some cases, a further test of stability can be made by growing the transformants on solid non-selective medium, harvesting the spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium.

VII. ANALYSIS FOR CBH1 NUCLEIC ACID CODING SEQUENCES AND/OR PROTEIN EXPRESSION

[0251] In order to evaluate the expression of a variant CBH I by a cell line that has been transformed with a variant CBH I-encoding nucleic acid construct, assays can be carried out at the protein level, the RNA level or by use of functional bioassays particular to cellobiohydrolase activity and/or production.

[0252] In one exemplary application of the variant cbh 1 nucleic acid and protein sequences described herein, a genetically modified strain of filamentous fungi, e.g., Trichoderma reesei, is engineered to produce an increased amount of CBH I. Such genetically modified filamentous fungi would be useful to produce a cellulase product with greater increased cellulolytic capacity. In one approach, this is accomplished by introducing the coding sequence for cbh 1 into a suitable host, e.g., a filamentous fungi such as Aspergillus niger.

[0253] Accordingly, the invention includes methods for expressing variant CBH I in a filamentous fungus or other suitable host by introducing an expression vector containing the DNA sequence encoding variant CBH I into cells of the filamentous fungus or other suitable host.

[0254] In another aspect, the invention includes methods for modifying the expression of CBH I in a filamentous fungus or other suitable host. Such modification includes a decrease or elimination in expression of the endogenous CBH.

[0255] In general, assays employed to analyze the expression of variant CBH I include, Northern blotting, dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction), or in situ hybridization, using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blotting and autoradiography.

[0256] In addition, the production and/or expression of variant CBH I may be measured in a sample directly, for example, by assays for cellobiohydrolase activity, expression and/or production. Such assays are described, for example, in Becker et al., Biochem J. (2001) 356:19-30 and Mitsuishi et al., FEBS (1990) 275:135-138, each of which is expressly incorporated by reference herein. The ability of CBH I to hydrolyze isolated soluble and insoluble substrates can be measured using assays described in Srisodsuk et al., J. Biotech. (1997) 57:49-57 and Nidetzky and Claeyssens Biotech. Bioeng. (1994) 44:961-966. Substrates useful for assaying cellobiohydrolase, endoglucanase or β-glucosidase activities include crystalline cellulose, filter paper, phosphoric acid swollen cellulose, cellooligosaccharides, methylumbelliferyl lactoside, methylumbelliferyl cellobioside, orthonitrophenyl lactoside, paranitrophenyl lactoside, orthonitrophenyl cellobioside, paranitrophenyl cellobioside.

[0257] In addition, protein expression, may be evaluated by immunological methods, such as immunohistochemical staining of cells, tissue sections or immunoassay of tissue culture medium, e.g., by Western blot or ELISA. Such immunoassays can be used to qualitatively and quantitatively evaluate expression of a CBH I variant. The details of such methods are known to those of skill in the art and many reagents for practicing such methods are commercially available.

[0258] A purified form of a variant CBH I may be used to produce either monoclonal or polyclonal antibodies specific to the expressed protein for use in various immunoassays. (See, e.g., Hu et al., Mol Cell Biol. vol. 11, no. 11, pp. 5792-5799, 1991). Exemplary assays include ELISA, competitive immunoassays, radioimmunoassays, Western blot, indirect immunofluorescent assays and the like. In general, commercially available antibodies and/or kits may be used for the quantitative immunoassay of the expression level of cellobiohydrolase proteins.

VIII. ISOLATION AND PURIFICATION OF RECOMBINANT CBH1 PROTEIN

[0259] In general, a variant CBH I protein produced in cell culture is secreted into the medium and may be purified or isolated, e.g., by removing unwanted components from the cell culture medium. However, in some cases, a variant CBH I protein may be produced in a cellular form necessitating recovery from a cell lysate. In such cases the variant CBH I protein is purified from the cells in which it was produced using techniques routinely employed by those of skill in the art. Examples include, but are not limited to, affinity chromatography (Tilbeurgh et al., FEBS Lett. 16:215, 1984), ion-exchange chromatographic methods (Goya) et al., Bioresource Technol. 36:37-50, 1991; Fliess et al., Eur. J. Appl. Microbiol. Biotechnol. 17:314-318, 1983; Bhikhabhai et al., J. Appl. Biochem. 6:336-345, 1984; Ellouz et al., J. Chromatography 396:307-317, 1987), including ion-exchange using materials with high resolution power (Medve et al., J. Chromatography A 808:153-165, 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatography A 865:123-128, 1999), and two-phase partitioning (Brumbauer, et al., Bioseparation 7:287-295, 1999).

[0260] Typically, the variant CBH I protein is fractionated to segregate proteins having selected properties, such as binding affinity to particular binding agents, e.g., antibodies or receptors; or which have a selected molecular weight range, or range of isoelectric points.

[0261] Once expression of a given variant CBH I protein is achieved, the CBH I protein thereby produced is purified from the cells or cell culture. Exemplary procedures suitable for such purification include the following: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, e.g., Sephadex G-75. Various methods of protein purification may be employed and such methods are known in the art and described e.g. in Deutscher, Methods in Enzymology, vol. 182, no. 57, pp. 779, 1990; Scopes, Methods Enzymol. 90: 479-91, 1982. The purification step(s) selected will depend, e.g., on the nature of the production process used and the particular protein produced.

IX. UTILITY OF CBH1 AND CBH1

[0262] It can be appreciated that the variant cbh nucleic acids, the variant CBH I protein and compositions comprising variant CBH I protein activity find utility in a wide variety applications, some of which are described below.

[0263] New and improved cellulase compositions that comprise varying amounts BG-type, EG-type and variant CBH-type cellulases find utility in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., "stone washing" or "biopolishing"), in compositions for degrading wood pulp into sugars (e.g., for bio-ethanol production), and/or in feed compositions. The isolation and characterization of cellulase of each type provides the ability to control the aspects of such compositions.

[0264] Variant (or mutant) CBHs with increased thermostability find uses in all of the above areas due to their ability to retain activity at elevated temperatures.

[0265] Variant (or mutant) CBHs with decreased thermostability find uses, for example, in areas where the enzyme activity is required to be neutralized at lower temperatures so that other enzymes that may be present are left unaffected. In addition, the enzymes may find utility in the limited conversion of cellulosics, for example, in controlling the degree of crystallinity or of cellulosic chain-length. After reaching the desired extent of conversion the saccharifying temperature can be raised above the survival temperature of the de-stabilized CBH I. As the CBH I activity is essential for hydrolysis of crystalline cellulose, conversion of crystalline cellulose will cease at the elevated temperature.

[0266] Variant (or mutant) CBHs with increased reversibility, i.e., enhanced refolding and retention of activity, also find use in similar areas. Depending upon the conditions of thermal inactivation, reversible denaturation can compete with, or dominate over, the irreversible process. Variants with increased reversibility would, under these conditions, exhibit increased resistance to thermal inactivation. Increased reversibility would also be of potential benefit in any process in which an inactivation event was followed by a treatment under non-inactivating conditions. For instance, in a Hybrid Hydrolysis and Fermentation (HHF) process for biomass conversion to ethanol, the biomass would first be incompletely saccharified by cellulases at elevated temperature (say 50° C. or higher), then the temperature would be dropped (to 30° C., for instance) to allow a fermentative organism to be introduced to convert the sugars to ethanol. If, upon decrease of process temperature, thermally inactivated cellulase reversibly re-folded and recovered activity then saccharification could continue to higher levels of conversion during the low temperature fermentation process.

[0267] In one approach, the cellulase of the invention finds utility in detergent compositions or in the treatment of fabrics to improve the feel and appearance.

[0268] Since the rate of hydrolysis of cellulosic products may be increased by using a transformant having at least one additional copy of the cbh gene inserted into the genome, products that contain cellulose or heteroglycans can be degraded at a faster rate and to a greater extent. Products made from cellulose such as paper, cotton, cellulosic diapers and the like can be degraded more efficiently in a landfill. Thus, the fermentation product obtainable from the transformants or the transformants alone may be used in compositions to help degrade by liquefaction a variety of cellulose products that add to the overcrowded landfills.

[0269] Separate saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and subsequently yeast strains convert glucose into ethanol. Simultaneous saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and, at the same time and in the same reactor, yeast strains convert glucose into ethanol. Thus, in another approach, the variant CBH type cellulase of the invention finds utility in the degradation of biomass to ethanol. Ethanol production from readily available sources of cellulose provides a stable, renewable fuel source.

[0270] Cellulose-based feedstocks are comprised of agricultural wastes, grasses and woods and other low-value biomass such as municipal waste (e.g., recycled paper, yard clippings, etc.). Ethanol may be produced from the fermentation of any of these cellulosic feedstocks. However, the cellulose must first be converted to sugars before there can be conversion to ethanol.

[0271] A large variety of feedstocks may be used with the inventive variant CBH and the one selected for use may depend on the region where the conversion is being done. For example, in the Midwestern United States agricultural wastes such as wheat straw, corn stover and bagasse may predominate while in California rice straw may predominate. However, it should be understood that any available cellulosic biomass may be used in any region.

[0272] A cellulase composition containing an enhanced amount of cellobiohydrolase finds utility in ethanol production. Ethanol from this process can be further used as an octane enhancer or directly as a fuel in lieu of gasoline which is advantageous because ethanol as a fuel source is more environmentally friendly than petroleum derived products. It is known that the use of ethanol will improve air quality and possibly reduce local ozone levels and smog. Moreover, utilization of ethanol in lieu of gasoline can be of strategic importance in buffering the impact of sudden shifts in non-renewable energy and petro-chemical supplies.

[0273] Ethanol can be produced via saccharification and fermentation processes from cellulosic biomass such as trees, herbaceous plants, municipal solid waste and agricultural and forestry residues. However, the ratio of individual cellulase enzymes within a naturally occurring cellulase mixture produced by a microbe may not be the most efficient for rapid conversion of cellulose in biomass to glucose. It is known that endoglucanases act to produce new cellulose chain ends which themselves are substrates for the action of cellobiohydrolases and thereby improve the efficiency of hydrolysis of the entire cellulase system. Therefore, the use of increased or optimized cellobiohydrolase activity may greatly enhance the production of ethanol.

[0274] Thus, the inventive cellobiohydrolase finds use in the hydrolysis of cellulose to its sugar components. In one embodiment, a variant cellobiohydrolase is added to the biomass prior to the addition of a fermentative organism. In a second embodiment, a variant cellobiohydrolase is added to the biomass at the same time as a fermentative organism. Optionally, there may be other cellulase components present in either embodiment.

[0275] In another embodiment the cellulosic feedstock may be pretreated. Pretreatment may be by elevated temperature and the addition of either of dilute acid, concentrated acid or dilute alkali solution. The pretreatment solution is added for a time sufficient to at least partially hydrolyze the hemicellulose components and then neutralized.

[0276] The major product of CBHI action on cellulose is cellobiose which is available for conversion to glucose by BG activity (for instance in a fungal cellulase product). Either by the pretreatment of the cellulosic biomass or by the enzymatic action on the biomass, other sugars, in addition to glucose and cellobiose, can be made available from the biomass. The hemi-cellulose content of the biomass can be converted (by hemi-cellulases) to sugars such as xylose, galactose, mannose and arabinose. Thus, in a biomass conversion process, enzymatic saccharification can produce sugars that are made available for biological or chemical conversions to other intermediates or end-products. Therefore, the sugars generated from biomass find use in a variety of processes in addition to the generation of ethanol. Examples of such conversions are fermentation of glucose to ethanol (as reviewed by M. E. Himmel et al. pp 2-45, in "Fuels and Chemicals from Biomass", ACS Symposium Series 666, ed B. C. Saha and J. Woodward, 1997) and other biological conversions of glucose to 2,5-diketo-D-gluconate (U.S. Pat. No. 6,599,722), lactic acid (R. Datta and S-P. Tsai pp 224-236, ibid), succinate (R. R. Gokarn, M. A. Eiteman and J. Sridhar pp 237-263, ibid), 1,3-propanediol (A-P. Zheng, H. Biebl and W-D. Deckwer pp 264-279, ibid), 2,3-butanediol (C. S. Gong, N. Cao and G. T. Tsao pp 280-293, ibid), and the chemical and biological conversions of xylose to xylitol (B. C. Saha and R. J. Bothast pp 307-319, ibid). See also, for example, WO 98/21339.

[0277] The detergent compositions of this invention may employ besides the cellulase composition (irrespective of the cellobiohydrolase content, i.e., cellobiohydrolase-free, substantially cellobiohydrolase-free, or cellobiohydrolase enhanced), a surfactant, including anionic, non-ionic and ampholytic surfactants, a hydrolase, building agents, bleaching agents, bluing agents and fluorescent dyes, caking inhibitors, solubilizers, cationic surfactants and the like. All of these components are known in the detergent art. The cellulase composition as described above can be added to the detergent composition either in a liquid diluent, in granules, in emulsions, in gels, in pastes, and the like. Such forms are well known to the skilled artisan. When a solid detergent composition is employed, the cellulase composition is preferably formulated as granules. Preferably, the granules can be formulated so as to contain a cellulase protecting agent. For a more thorough discussion, see U.S. Pat. No. 6,162,782 entitled "Detergent compositions containing cellulase compositions deficient in CBH I type components," which is incorporated herein by reference.

[0278] Preferably the cellulase compositions are employed from about 0.00005 weight percent to about 5 weight percent relative to the total detergent composition. More preferably, the cellulase compositions are employed from about 0.0002 weight percent to about 2 weight percent relative to the total detergent composition.

[0279] In addition the variant cbh I nucleic acid sequence finds utility in the identification and characterization of related nucleic acid sequences. A number of techniques useful for determining (predicting or confirming) the function of related genes or gene products include, but are not limited to, (A) DNA/RNA analysis, such as (1) overexpression, ectopic expression, and expression in other species; (2) gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS, see Baulcombe, 100 Years of Virology, Calisher and Horzinek eds., Springer-Verlag, New York, N.Y. 15:189-201, 1999); (3) analysis of the methylation status of the gene, especially flanking regulatory regions; and (4) in situ hybridization; (B) gene product analysis such as (1) recombinant protein expression; (2) antisera production, (3) immunolocalization; (4) biochemical assays for catalytic or other activity; (5) phosphorylation status; and (6) interaction with other proteins via yeast two-hybrid analysis; (C) pathway analysis, such as placing a gene or gene product within a particular biochemical or signaling pathway based on its overexpression phenotype or by sequence homology with related genes; and (D) other analyses which may also be performed to determine or confirm the participation of the isolated gene and its product in a particular metabolic or signaling pathway, and help determine gene function.

[0280] All patents, patent applications, articles and publications mentioned herein, are hereby expressly incorporated herein by reference.

EXAMPLES

[0281] The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein.

Example 1

Alignment of Known Cel7A Cellulases

[0282] The choice of several of the mutations was determined by first aligning Hypocrea jecorina Cel7A to its 41 family members using structural information and a modeling program. The alignment of the primary amino acid sequence of all 42 family members is shown in FIG. 8 (SEQ ID NOs:32-73); with the consensus shown in SEQ ID NO:74.

[0283] For four of the members (i.e., 20VW.1, 1A39, 6CEL and 1EG1.1), the crystal structure had been previously determined. The 4 aligned proteins for which there were published structures had their alignment locked for all residues whose backbone atoms were within a specific RMS deviation (RMS less than or equal to 2.0 A). The tertiary structural alignment of the four sequences was performed using MOE version 2001.01 by Chemical Computing Group, Montreal Canada. The overlapping structural elements were used to freeze the primary structures of the four sequences. The remaining 38 sequences then had their primary amino acid structure aligned with the frozen four using MOE with secondary structure prediction on and other parameters set to their default settings.

[0284] Based on the alignments, various single and multiple amino acid mutations were made in the protein by site mutagenesis.

[0285] Single amino acid mutations were based on the following rationale (see also Table 1): After examining the conservation of amino acids between the homologues, sites were picked in the H. jecorina sequence where a statistical preference for another amino acid was seen amongst the other 41 sequences (e.g.: at position 77 the Ala, only present in H. jecorina and 3 other homologues, was changed to Asp, present in 22 others). The effect of each substitution on the structure was then modeled.

TABLE-US-00001 TABLE 1 Cel7A Variants and Rationale for Change Cel7A Variants and Rationale for Change Tm ΔTm Wild Type H. jecorina 62.5 (4)A77D(22) 3 possible H-bonds to Q7 and I80 62.2 -0.3 (7)S113D(18) numerous new H-bonds to backbone 62.8 0.3 to stabilize turn (8)L225F(13) better internal packing 61.6 -0.9 (5)L288F(17) better internal packing 62.4 -0.1 (1)A299E(24) extra ligand to cobalt atom observed 61.2 -1.3 in crystal structure (4)N301K(11) salt bridges to E295 and E325 63.5 1.0 (5)T356L(20) better internal packing 62.6 0.1 (2)G430F(17) better surface packing 61.7 -0.8

[0286] Multiple amino acid mutations were based on a desire to affect the stability, processivity, and product inhibition of the enzyme. The following multiple site changes in the H. jecorina sequence were constructed:

[0287] 1) Thr 246 Cys+Tyr 371 Cys

[0288] 2) Thr 246 Ala+Arg 251 Ala+Tyr 252 Ala

[0289] 3) Thr 380 Gly+Tyr 381 Asp+Arg 394 Ala+deletion of Residues 382 to 393, inclusive

[0290] 4) Thr 380 Gly+Tyr 381 Asp+Arg 394 Ala

[0291] 5) Tyr 252 Gln+Asp 259 Trp+Ser 342 Tyr

[0292] The T246A/R251A/Y252A and the other triple+deletion mutant are both predicted to decrease the product inhibition of the enzyme. The Thr246Cys+Tyr371 Cys is predicted to increase the stability of the enzyme and increase the processitivity of it. The D259W/Y252Q/S342Y variant is predicted to affect the product inhibition of the enzyme.

[0293] Other single and multiple mutations were constructed using methods well known in the art (see references above) and are presented in Table 2.

TABLE-US-00002 TABLE 2 H. jecorina CBH I variants Mutations S8P N49S A68T A77D N89D S92T S113N S113D L225F P227A P227L D249K T255P D257E S279N L288F E295K S297T A299E N301K T332K T332Y T332H T356L F338Y V393G G430F T41I (plus deletion of Thr @ 445) V403D/T462I S196T/S411F E295K/S398T A112E/T226A T246C/Y371C G22D/S278P/T296P S8P/N103I/S113N S113T/T255P/K286M P227L/E325K/Q487L P227T/T484S/F352L T246A/R251A/Y252A T380G/Y381D/R394A Y252Q/D259W/S342Y A68T/G440R/P491L Q17L/E193V/M213I/F352L S8P/N49S/A68T/S113N A112E/P227L/S278P/T296P S8P/N49S/A68T/N103I/S113N S8P/N49S/A68T/S278P/T296P G22D/N49S/A68T/S278P/T296P G22D/N103I/S113N/S278P/T296P S8P/N49S/A68T/S113N/P227L S8P/N49S/A68T/A112E/T226A S8P/N49S/A68T/A112E/P227L T41I/A112E/P227L/S278P/T296P S8P/T41I/N49S/A68T/S113N/P227L S8P/T41I/N49S/A68T/A112E/P227L G22D/N49S/A68T/P227L/S278P/T296P G22D/N49S/A68T/N103I/S113N/S278P/T296P G22D/N49S/A68T/N103I/S113N/P227L/S278P/T296P G22D/N49S/A68T/N103I/A112E/P227L/S278P/T296P G22D/N49S/N64D/A68T/N103I/S113N/S278P/T296P S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F S8P/G22D/T41I/N49S/A68T/N103I/S113N/S278P/T296P S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/N301R S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/T296P/N301R S8P/T41I/N49S/S57N/A68T/S113N/P227L/D249K/S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/ S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/ S278P/T296P/N301R/E325K/S411F S8P/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/ T296P/N301R/E325K/V403D/S411F/T462I S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/ S278P/T296P/N301R/E325K/V403D/S411F/T462I

Example 2

Cloning and Expression of CBHI Variants in H. jecorina

[0294] A. Construction of the H. jecorina general-purpose expression plasmid-PTEX.

[0295] The plasmid, pTEX was constructed following the methods of Sambrook et al. (1989), supra, and is illustrated in FIG. 7. This plasmid has been designed as a multi-purpose expression vector for use in the filamentous fungus Trichoderma longibrachiatum. The expression cassette has several unique features that make it useful for this function. Transcription is regulated using the strong CBH I gene promoter and terminator sequences for T. longibrachiatum. Between the CBHI promoter and terminator there are unique PmeI and SstI restriction sites that are used to insert the gene to be expressed. The T. longibrachiatum pyr4 selectable marker gene has been inserted into the CBHI terminator and the whole expression cassette (CBHI promoter-insertion sites-CBHI terminator-pyr4 gene-CBHI terminator) can be excised utilizing the unique NotI restriction site or the unique NotI and NheI restriction sites.

[0296] This vector is based on the bacterial vector, pSL1180 (Pharmacia Inc., Piscataway, N.J.), which is a PUC-type vector with an extended multiple cloning site. One skilled in the art would be able to construct this vector based on the flow diagram illustrated in FIG. 7.

[0297] The vector pTrex2L was constructed from pTrex2, a derivative of pTEX. The sequence for pTrex2 is given in FIG. 6 (SEQ ID NO:31).

[0298] The exact plasmid used is not that important as long as the variant protein is expressed at a useful level. However, maximizing the expression level by forcing integration at the cbh1 locus is advantageous.

B. Cloning

[0299] Using methods known in the art a skilled person can clone the desired CBH I variant into an appropriate vector. As noted above, the exact plasmid used is not that important as long as the variant protein is expressed at a useful level. The following description of the preparation of one of the inventive variant CBH I enzymes can be utilized to prepare any of the inventive variants described herein.

[0300] The variant cbh 1 genes were cloned into the pTrex2L vector.

[0301] Construction of plasmid pTrex2L was done as follows: The 6 nucleotides between the unique Sac II and Asc I sites of pTrex2 (SEQ ID NO:78) were replaced with a synthetic linker (SEQ ID NO:79) containing a BstE II and BamH I sites to produce plasmid Trex2L. The complementary synthetic linkers

TABLE-US-00003 (SEQ ID NO: 75) 21-mer synthetic oligo CBHlink1+: GGTTTGGATCCGGTCACCAGG and (SEQ ID NO: 76) 27-mer synthetic oligo CBHlink-: CGCGCCTGGTGACCGGATCCAAACCGC

were annealed.

[0302] The pTrex2 was digested with Sac II and Asc I. The annealed linker (SEQ ID NO:80) was then ligated into pTrex2 to create pTrex2L. The plasmid was then digested with an appropriate restriction enzyme(s) and a wild type CBH I gene was ligated into the plasmid.

[0303] Primers were used to introduce the desired mutations into the wild-type gene. It will be understood that any method that results in the introduction of a desired alteration or mutation in the gene may be used. Synthetic DNA primers were used as PCR templates for mutant constructions. It is well within the knowledge of the skilled artisan to design the primers based on the desired mutation to be introduced.

[0304] The mutagenic templates were extended and made double stranded by PCR using the synthetic DNA oligonucleotides. After 25 PCR cycles the final product was primarily a 58 by double stranded product comprising the desired mutation. The mutagenic fragments were subsequently attached to wild-type CBH I fragments and ligated into the plasmid using standard techniques.

C. Transformation and Expression

[0305] The prepared vector for the desired variant was transformed into the uridine auxotroph version of the double or quad deleted Trichoderma strains (see Table 3; see also U.S. Pat. Nos. 5,861,271 and 5,650,322) and stable transformants were identified.

TABLE-US-00004 TABLE 3 Transformation/Expression strain CBH I Variant Expression Strain A77D quad-delete strain (1A52) S113D double-delete strain L225F double-delete strain L288F double-delete strain A299E quad-delete strain (1A52) N301K quad-delete strain (1A52) T356L double-delete strain G430F quad-delete strain (1A52) T246C/Y371C quad-delete strain (1A52) T246A/R251A/Y252A quad-delete strain (1A52) Y252Q/D259W/S342Y quad-delete strain (1A52) T380G/Y381D/R394A quad-delete strain (1A52) T380G/Y381D/R394A quad-delete strain (1A52) plus deletion of 382-393 "double-delete" (Δ CBHI & Δ CBHII) and the "quad-delete" (Δ CBHI & Δ CBHII, Δ EGI & Δ EGII) T. reesei host strains

[0306] To select which transformants expressed variant CBH I, DNA was isolated from strains following growth on Vogels+1% glucose and Southern blot experiments performed using an isolated DNA fragment containing only the variant CBH I. Transformants were isolated having a copy of the variant CBH I expression cassette integrated into the genome of the host cell. Total mRNA was isolated from the strains following growth for 1 day on Vogels+1% lactose. The mRNA was subjected to Northern analysis using the variant CBH I coding region as a probe. Transformants expressing variant CBH I mRNA were identified.

[0307] One may obtain any other novel variant CBH I cellulases or derivative thereof by employing the methods described above.

Example 3

Expression of CBH1 Variants in A. niger

[0308] The PCR fragments were obtained using the following primers and protocols

[0309] The following DNA primers were constructed for use in amplification of homologous CBH1 genes from genomic DNA's isolated from various microorganisms. All symbols used herein for protein and DNA sequences correspond to IUPAC IUB Biochemical Nomenclature Commission codes.

[0310] Homologous 5' (FRG192) and 3' (FRG193) primers were developed based on the sequence of CBH1 from Trichoderma reesei. Both primers contained Gateway cloning sequences from Invitrogen® at the 5' of the primer. Primer FRG192 contained attB1 sequence and primer FRG193 contained attB2 sequence.

TABLE-US-00005 (SEQ ID NO: 3) Sequence of FRG192 without the attB1: ATGTATCGGAAGTTGGCCG (signal sequence of CBH1 H. jecorina) (SEQ ID NO: 4) Sequence of FRG193 without the attB2: TTACAGGCACTGAGAGTAG (cellulose binding module of CBH1 H. jecorina)

[0311] The H. jecorina CBH I cDNA clone served as template.

[0312] PCR conditions were as follows: 10 μL of 10× reaction buffer (10× reaction buffer comprising 100 mM Tris HCl, pH 8-8.5; 250 mM KCl; 50 mM (NH₄)₂SO₄; 20 mM MgSO₄); 0.2 mM each of dATP, dTTP, dGTP, dCTP (final concentration), 1 μL of 100 ng/μL genomic DNA, 0.5 μL of PWO polymerase (Boehringer Mannheim, Cat #1644-947) at 1 unit per μL, 0.2 μM of each primer, FRG192 and FRG193, (final concentration), 4 μl DMSO and water to 100 μL.

[0313] Various sites in H. jecorina CBH1 may be involved in the thermostability of the variants and the H. jecorina CBH1 gene was therefore subjected to mutagenesis.

[0314] The fragments encoding the variants were purified from an agarose gel using the Qiagen Gel extraction KIT. The purified fragments were used to perform a clonase reaction with the pDONR®201 vector from Invitrogen® using the Gateway® Technology instruction manual (version C) from Invitrogen®, hereby incorporated by reference herein. Genes were then transferred from this ENTRY vector to the destination vector (pRAXdes2) to obtain the expression vector pRAXCBH1.

[0315] Cells were transformed with an expression vector comprising a variant CBH I cellulase encoding nucleic acid. The constructs were transformed into A. niger var. awamori according to the method described by Cao et al (Cao Q-N, Stubbs M, Ngo KQP, Ward M, Cunningham A, Pai E F, Tu G-C and Hofmann T (2000) Penicillopepsin-JT2 a recombinant enzyme from Penicillium janthinellum and contribution of a hydrogen bond in subsite S3 to kcat Protein Science 9:991-1001).

[0316] Transformants were streaked on minimal medium plates (Ballance D J, Buxton F P, and Turner G (1983) Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa Biochem Biophys Res Commun 112:284-289) and grown for 4 days at 30° C. Spores were collected using methods well known in the art (See fgsc.net/fgn48/Kaminskyj.htm). A. nidulans conidia are harvested in water (by rubbing the surface of a conidiating culture with a sterile bent glass rod to dislodge the spores) and can be stored for weeks to months at 4° C. without a serious loss of viability. However, freshly harvested spores germinate more reproducibly. For long-term storage, spores can be stored in 50% glycerol at -20° C., or in 15-20% glycerol at -80° C. Glycerol is more easily pipetted as an 80% solution in water. 800 μl of aqueous conidial suspension (as made for 4° C. storage) added to 200 μl 80% glycerol is used for a -80° C. stock; 400 μl suspension added to 600 μl 80% glycerol is used for a -20° C. stock. Vortex before freezing. For mutant collections, small pieces of conidiating cultures can be excised and placed in 20% glycerol, vortexed, and frozen as -80° C. stocks. In our case we store them in 50% glycerol at -80° C.

[0317] A. niger var awamori transformants were grown on minimal medium lacking uridine (Ballance et al. 1983). Transformants were screened for cellulase activity by inoculating 1 cm² of spore suspension from the sporulated grown agar plate into 100 ml shake flasks for 3 days at 37° C. as described by Cao et al. (2000).

[0318] The CBHI activity assay is based on the hydrolysis of the nonfluorescent 4-methylumbelliferyl-β-lactoside to the products lactose and 7-hydroxy-4-methylcoumarin, the latter product is responsible for the fluorescent signal. Pipette 170 μl 50 mM NaAc buffer pH 4.5 in a 96-well microtiter plate (MTP) (Greiner, Fluotrac 200, art. nr. 655076) suitable for fluorescence. Add 10 μl of supernatant and then add 10 μl of MUL (1 mM 4-methylumbelliferyl-R-lactoside (MUL) in milliQ water) and put the MTP in the Fluostar Galaxy (BMG Labtechnologies; D-77656 Offenburg). Measure the kinetics for 16 min. (8 cycles of 120s each) using λ₃₂₀ nm (excitation) and λ₄₆₀ nm (emission) at 50° C. Supernatents having CBH activity were then subjected to Hydrophobic Interaction Chromatography.

Example 4

Stability of CBH 1 Variants

[0319] CBH I cellulase variants were cloned and expressed as above (see Examples 2 and 3). Cel7A wild type and variants were then purified from cell-free supernatants of these cultures by column chromatography. Proteins were purified using hydrophobic interaction chromatography (HIC). Columns were run on a BioCAD® Sprint Perfusion Chromatography System using Poros® 20 HP2 resin both made by Applied Biosystems.

[0320] HIC columns were equilibrated with 5 column volumes of 0.020 M sodium phosphate, 0.5 M ammonium sulfate at pH 6.8. Ammonium sulfate was added to the supernatants to a final concentration of approximately 0.5 M and the pH was adjusted to 6.8. After filtration, the supernatant was loaded onto the column. After loading, the column was washed with 10 column volumes of equilibration buffer and then eluted with a 10 column volume gradient from 0.5 M ammonium sulfate to zero ammonium sulfate in 0.02 M sodium phosphate pH 6.8. Cel7A eluted approximately mid-gradient. Fractions were collected and pooled on the basis of reduced, SDS-PAGE gel analysis.

[0321] The melting points were determined according to the methods of Luo, et al., Biochemistry 34:10669 and Gloss, et al., Biochemistry 36:5612. See also Sandgren at al. (2003) Protein Science 12(4) pp 848.

[0322] Data was collected on the Aviv 215 circular dichroism spectrophotometer. The native spectra of the variants between 210 and 260 nanometers were taken at 25° C. Buffer conditions were 50 mM Bis Tris Propane/50 mM ammonium acetate/glacial acetic acid at pH 5.5. The protein concentration was kept between 0.25 and 0.5 mgs/mL. After determining the optimal wavelength to monitor unfolding, the samples were thermally denatured by ramping the temperature from 25° C. to 75° C. under the same buffer conditions. Data was collected for 5 seconds every 2 degrees. Partially reversible unfolding was monitored at 230 nanometers in a 0.1 centimeter path length cell. While at 75° C., an unfolded spectra was collected as described above. The sample was then cooled to 25° C. to collect a refolded spectra. The difference between the three spectra at 230 nm was used to assess the variants reversibility.

[0323] The thermal denaturation profiles are shown in FIGS. 9A and 9B for wildtype CBH I and various variant CBH I's. See also Table 4.

TABLE-US-00006 TABLE 4 Thermal Stability of Variant CBH I cellulases delta % rev H. jecorina CBH I Residue Substitution Tm Tm 230 nm Wild type 62.5 23 S8P 63.1 0.6 N49S 63.7 1.2 A68T 63.7 1.2 32 A77D 62.2 -0.3 N89D 63.6 1.1 50 S92T 64.4 1.9 25 S113D 62.8 0.3 S113N 64.0 1.5 L225F 61.6 -0.9 P227A 64.8 2.3 49 P227L 65.2 2.7 45 D249K 64.0 1.5 39 T255P 64.4 1.9 35 S279N 62.4 -0.1 ~95 E295K 64.0 1.5 ~95 T332K 63.3 0.8 37 T332Y 63.3 0.8 37 T332H 62.7 0.2 64 F338Y 60.8 -1.7 ~95 G430F 61.7 -0.8 L288F 62.4 -0.1 A299E 61.2 -1.3 N301K 63.5 1.0 T356L 62.6 0.1 D257E 61.8 -0.7 45 V393G 61.7 -0.8 43 S297T 63.3 0.8 31 T41I plus deletion @ T445 64.2 1.7 T246C/Y371C 65.0 2.5 S196T/S411F 65.3 2.8 27 E295K/S398T 63.9 1.4 36 V403D/T462I 64.5 2 53 A112E/T226A 63.5 1.0 A68T/G440R/P491L 63.1 0.6 32 G22D/S278P/T296P 63.6 1.1 T246A/R251A/Y252A 63.5 1.0 T380G/Y381D/R394A 58.1 -4.4 Y252Q/D259W/S342Y 59.9 -2.6 50 S113T/T255P/K286M 63.8 1.3 16 P227L/E325K/Q487L 64.5 2.0 22 P227T/T484S/F352L 64.2 1.7 45 Q17L/E193V/M213I/F352L 64.0 1.5 34 S8P/N49S/A68T/S113N 64.5 2.0 90 S8P/N49S/A68T/S113N/P227L 66.0 3.5 86 T41I/A112E/P227L/S278P/T296P 66.1 3.6 48 S8P/N49S/A68T/A112E/T226A 64.6 2.1 46 S8P/N49S/A68T/A112E/P227L 65.2 2.7 32 S8P/T41I/N49S/A68T/A112E/P227L 67.6 5.1 40 G22D/N49S/A68T/P227L/S278P/T296P 65.9 3.4 26 G22D/N49S/A68T/N103I/S113N/P227L/S278P/ 65.3 2.8 72 T296P G22D/N49S/A68T/N103I/A112E/P227L/S278P/ 65.1 2.6 20 T296P G22D/N49S/N64D/A68T/N103I/S113N/S278P/ 61.4 -1.1 75 T296P S8P/G22D/T41I/N49S/A68T/N103I/S113N/ 68.8 6.3 56 P227L/S278P/T296P S8P/G22D/T41I/N49S/A68T/N103I/S113N/ 69.0 6.5 71 P227L/D249K/S278P/T296P S8P/G22D/T41I/N49S/A68T/N103I/S113N/ 68.7 6.2 70 P227L/S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/N103I/S113N/ 68.8 6.3 74 P227L/D249K/S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/S113N/P227L/ 69.9 7.4 88 D249K/S278P/T296P/N301R S8P/T41I/N49S/S57N/A68T/S113N/P227L/ 68.9 6.4 ~100 D249K/S278P/T296P/N301R S8P/G22D/T41I/N49S/A68T/S113N/P227L/ 68.7 6.2 92 D249K/S278P/N301R S8P/T41I/N49S/A68T/S92T/S113N/P227L/ 68.8 6.3 ~100 D249K/V403D/T462I S8P/G22D/T41I/N49S/A68T/S92T/S113N/ 68.5 6.0 ~100 P227L/D249K/V403D/T462I S8P/T41I/N49S/A68T/S92T/S113N/P227L/ 68.6 6.1 ~100 D249K/S411F S8P/G22D/T41I/N49S/A68T/S92T/S113N/ 69.5 7.0 ~100 P227L/D249K/S411F S8P/G22D/T41I/N49S/A68T/S92T/S113N/ 70.7 8.2 ~100 S196T/P227L/D249K/T255P/S278P/T296P/ N301R/E325K/S411F S8P/T41I/N49S/A68T/S92T/S113N/S196T/ 71.0 8.5 ~100 P227L/D249K/T255P/S278P/T296P/N301R/ E325K/V403D/S411F/T462I S8P/G22D/T41I/N49S/A68T/S92T/S113N/ 70.9 8.4 ~100 S196T/P227L/D249K/T255P/S278P/T296P/ N301R/E325K/V403D/S411F/T462I

[0324] Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

Sequence CWU 1

1

7711491DNAHypocrea jecorina 1cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 14912497PRTHypocrea jecorina 2Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu319DNAArtificial Sequenceprimer 3atgtatcgga agttggccg 19419DNAArtificial Sequenceprimer 4ttacaggcac tgagagtag 1951491DNAHypocrea jecorina 5cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg cttttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 14916497PRTHypocrea jecorina 6Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Phe Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu71491DNAHypocrea jecorina 7cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcggacg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 14918497PRTHypocrea jecorina 8Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Asp Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu91491DNAHypocrea jecorina 9cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccga ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc

ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149110497PRTHypocrea jecorina 10Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Asp Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu111491DNAHypocrea jecorina 11cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa atttaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149112497PRTHypocrea jecorina 12Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Phe 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu131491DNAHypocrea jecorina 13cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgagatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149114497PRTHypocrea jecorina 14Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Glu Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu151491DNAHypocrea jecorina 15cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aagcgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149116497PRTHypocrea jecorina 16Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145

150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Lys Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu171491DNAHypocrea jecorina 17cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctctctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149118497PRTHypocrea jecorina 18Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Leu Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu191491DNAHypocrea jecorina 19cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccttc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149120510PRTHypocrea jecorina 20Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 35 40 45Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp 50 55 60Asn Glu Thr Cys Ile Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr65 70 75 80Ala Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly 85 90 95Phe Val Thr Gln Ser Ala Ile Gln Lys Asn Val Gly Ala Arg Leu Tyr 100 105 110Leu Met Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn 115 120 125Glu Phe Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn 130 135 140Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys145 150 155 160Tyr Pro Thr Asn Thr Ala Gly Ala Lys Tyr Ile Gly Thr Gly Tyr Cys 165 170 175Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn 180 185 190Val Glu Gly Trp Glu Pro Ser Ser Asn Asn Ala Asn Ile Thr Gly Ile 195 200 205Gly Gly His Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn 210 215 220Ser Ile Ser Glu Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Ile225 230 235 240Gln Glu Ile Cys Glu Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn 245 250 255Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr 260 265 270Arg Ile Leu Gly Asn Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr 275 280 285Leu Asp Thr Thr Lys Lys Leu Thr Val Val Thr Gln Phe Glu Thr Ser 290 295 300Gly Ala Ile Ile Asn Arg Tyr Tyr Val Gln Asn Gly Val Thr Phe Gln305 310 315 320Gln Pro Asn Ala Glu Leu Gly Ser Tyr Ser Gly Asn Glu Leu Asn Asp 325 330 335Asp Tyr Cys Thr Ala Glu Ile Glu Ala Glu Phe Gly Gly Ser Ser Phe 340 345 350Ser Asp Lys Gly Gly Leu Thr Gln Phe Lys Lys Ala Thr Ser Gly Gly 355 360 365Met Val Leu Val Met Ser Leu Trp Ile Asp Asp Tyr Tyr Ala Asn Met 370 375 380Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr Pro385 390 395 400Gly Ala Val Arg Gly Ser Cys Ser Thr Ser Ile Ser Gly Val Pro Ala 405 410 415Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val Thr Phe Ser Asn Ile 420 425 430Lys Phe Gly Pro Ile Gly Ser Thr Phe Asn Pro Ser Gly Ile Gly Asn 435 440 445Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr Arg Arg Pro Ala 450 455 460Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser His Tyr Gly Ile465 470 475 480Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly 485 490 495Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu 500 505 510211491DNAHypocrea jecorina 21cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggatgtta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tgcgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149122497PRTHypocrea jecorina 22Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45 Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Cys Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys

Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Cys Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu231491DNAHypocrea jecorina 23cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggagctta ctccgataac gcagctggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149124497PRTHypocrea jecorina 24Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Ala Tyr Ser Asp Asn Ala Ala Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu251491DNAHypocrea jecorina 25cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccggc 1140gacccgacaa acgagacctc ctccacaccc ggtgccgtgg ccggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149126497PRTHypocrea jecorina 26Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Gly Asp Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Ala Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu271455DNAHypocrea jecorina 27cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agatatggcg gcacttgcga tcccgatggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctctttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccggc 1140gacgccggaa gctgctccac cagctccggt gtccctgctc aggtcgaatc tcagtctccc 1200aacgccaagg tcaccttctc caacatcaag ttcggaccca ttggcagcac cggcaaccct 1260agcggcggca accctcccgg cggaaacccg cctggcacca ccaccacccg ccgcccagcc 1320actaccactg gaagctctcc cggacctacc cagtctcact acggccagtg cggcggtatt 1380ggctacagcg gccccacggt ctgcgccagc ggcacaactt gccaggtcct gaacccttac 1440tactctcagt gcctg 145528486PRTHypocrea jecorina 28Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys 245 250 255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Gly Asp Ala Gly Ser 370 375 380Cys Ser Thr Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro385 390 395 400Asn Ala Lys Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser 405 410 415Thr Gly Asn Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly 420 425 430Thr Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro 435 440 445Gly Pro Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser 450 455 460Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro465 470 475 480Tyr Tyr Ser Gln Cys Leu 485291491DNAHypocrea jecorina 29cagtcggcct gcactctcca atcggagact cacccgcctc tgacatggca gaaatgctcg 60tctggtggca cttgcactca acagacaggc tccgtggtca tcgacgccaa ctggcgctgg 120actcacgcta cgaacagcag cacgaactgc tacgatggca acacttggag ctcgacccta 180tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg acggtgccgc ctacgcgtcc 240acgtacggag ttaccacgag cggtaacagc ctctccattg gctttgtcac ccagtctgcg 300cagaagaacg ttggcgctcg cctttacctt atggcgagcg acacgaccta ccaggaattc 360accctgcttg gcaacgagtt ctctttcgat gttgatgttt cgcagctgcc gtgcggcttg 420aacggagctc tctacttcgt gtccatggac gcggatggtg gcgtgagcaa

gtatcccacc 480aacaccgctg gcgccaagta cggcacgggg tactgtgaca gccagtgtcc ccgcgatctg 540aagttcatca atggccaggc caacgttgag ggctgggagc cgtcatccaa caacgcgaac 600acgggcattg gaggacacgg aagctgctgc tctgagatgg atatctggga ggccaactcc 660atctccgagg ctcttacccc ccacccttgc acgactgtcg gccaggagat ctgcgagggt 720gatgggtgcg gcggaactta ctccgataac agacagggcg gcacttgcga tccctggggc 780tgcgactgga acccataccg cctgggcaac accagcttct acggccctgg ctcaagcttt 840accctcgata ccaccaagaa attgaccgtt gtcacccagt tcgagacgtc gggtgccatc 900aaccgatact atgtccagaa tggcgtcact ttccagcagc ccaacgccga gcttggtagt 960tactctggca acgagctcaa cgatgattac tgcacagctg aggaggcaga attcggcgga 1020tcctatttct cagacaaggg cggcctgact cagttcaaga aggctacctc tggcggcatg 1080gttctggtca tgagtctgtg ggatgattac tacgccaaca tgctgtggct ggactccacc 1140tacccgacaa acgagacctc ctccacaccc ggtgccgtgc gcggaagctg ctccaccagc 1200tccggtgtcc ctgctcaggt cgaatctcag tctcccaacg ccaaggtcac cttctccaac 1260atcaagttcg gacccattgg cagcaccggc aaccctagcg gcggcaaccc tcccggcgga 1320aacccgcctg gcaccaccac cacccgccgc ccagccacta ccactggaag ctctcccgga 1380cctacccagt ctcactacgg ccagtgcggc ggtattggct acagcggccc cacggtctgc 1440gccagcggca caacttgcca ggtcctgaac ccttactact ctcagtgcct g 149130497PRTHypocrea jecorina 30Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser65 70 75 80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val 85 90 95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala 100 105 110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser 115 120 125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu 130 135 140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr145 150 155 160Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 165 170 175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp 180 185 190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser 195 200 205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala 210 215 220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly225 230 235 240Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Gln Gly Gly Thr Cys 245 250 255Asp Pro Trp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser 260 265 270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu 275 280 285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr 290 295 300Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser305 310 315 320Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala 325 330 335Glu Phe Gly Gly Ser Tyr Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe 340 345 350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp 355 360 365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 370 375 380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser385 390 395 400Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val 405 410 415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro 420 425 430Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr 435 440 445Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 450 455 460His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys465 470 475 480Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 485 490 495Leu318970DNAArtificial SequencepTrex2 vector 31aagcttaagg tgcacggccc acgtggccac tagtacttct cgagctctgt acatgtccgg 60tcgcgacgta cgcgtatcga tggcgccagc tgcaggcggc cgcctgcagc cacttgcagt 120cccgtggaat tctcacggtg aatgtaggcc ttttgtaggg taggaattgt cactcaagca 180cccccaacct ccattacgcc tcccccatag agttcccaat cagtgagtca tggcactgtt 240ctcaaataga ttggggagaa gttgacttcc gcccagagct gaaggtcgca caaccgcatg 300atatagggtc ggcaacggca aaaaagcacg tggctcaccg aaaagcaaga tgtttgcgat 360ctaacatcca ggaacctgga tacatccatc atcacgcacg accactttga tctgctggta 420aactcgtatt cgccctaaac cgaagtgcgt ggtaaatcta cacgtgggcc cctttcggta 480tactgcgtgt gtcttctcta ggtgccattc ttttcccttc ctctagtgtt gaattgtttg 540tgttggagtc cgagctgtaa ctacctctga atctctggag aatggtggac taacgactac 600cgtgcacctg catcatgtat ataatagtga tcctgagaag gggggtttgg agcaatgtgg 660gactttgatg gtcatcaaac aaagaacgaa gacgcctctt ttgcaaagtt ttgtttcggc 720tacggtgaag aactggatac ttgttgtgtc ttctgtgtat ttttgtggca acaagaggcc 780agagacaatc tattcaaaca ccaagcttgc tcttttgagc tacaagaacc tgtggggtat 840atatctagag ttgtgaagtc ggtaatcccg ctgtatagta atacgagtcg catctaaata 900ctccgaagct gctgcgaacc cggagaatcg agatgtgctg gaaagcttct agcgagcggc 960taaattagca tgaaaggcta tgagaaattc tggagacggc ttgttgaatc atggcgttcc 1020attcttcgac aagcaaagcg ttccgtcgca gtagcaggca ctcattcccg aaaaaactcg 1080gagattccta agtagcgatg gaaccggaat aatataatag gcaatacatt gagttgcctc 1140gacggttgca atgcaggggt actgagcttg gacataactg ttccgtaccc cacctcttct 1200caacctttgg cgtttccctg attcagcgta cccgtacaag tcgtaatcac tattaaccca 1260gactgaccgg acgtgttttg cccttcattt ggagaaataa tgtcattgcg atgtgtaatt 1320tgcctgcttg accgactggg gctgttcgaa gcccgaatgt aggattgtta tccgaactct 1380gctcgtagag gcatgttgtg aatctgtgtc gggcaggaca cgcctcgaag gttcacggca 1440agggaaacca ccgatagcag tgtctagtag caacctgtaa agccgcaatg cagcatcact 1500ggaaaataca aaccaatggc taaaagtaca taagttaatg cctaaagaag tcatatacca 1560gcggctaata attgtacaat caagtggcta aacgtaccgt aatttgccaa cggcttgtgg 1620ggttgcagaa gcaacggcaa agccccactt ccccacgttt gtttcttcac tcagtccaat 1680ctcagctggt gatcccccaa ttgggtcgct tgtttgttcc ggtgaagtga aagaagacag 1740aggtaagaat gtctgactcg gagcgttttg catacaacca agggcagtga tggaagacag 1800tgaaatgttg acattcaagg agtatttagc cagggatgct tgagtgtatc gtgtaaggag 1860gtttgtctgc cgatacgacg aatactgtat agtcacttct gatgaagtgg tccatattga 1920aatgtaagtc ggcactgaac aggcaaaaga ttgagttgaa actgcctaag atctcgggcc 1980ctcgggcctt cggcctttgg gtgtacatgt ttgtgctccg ggcaaatgca aagtgtggta 2040ggatcgaaca cactgctgcc tttaccaagc agctgagggt atgtgatagg caaatgttca 2100ggggccactg catggtttcg aatagaaaga gaagcttagc caagaacaat agccgataaa 2160gatagcctca ttaaacggaa tgagctagta ggcaaagtca gcgaatgtgt atatataaag 2220gttcgaggtc cgtgcctccc tcatgctctc cccatctact catcaactca gatcctccag 2280gagacttgta caccatcttt tgaggcacag aaacccaata gtcaaccgcg gtttaggcgc 2340gccagctccg tgcgaaagcc tgacgcaccg gtagattctt ggtgagcccg tatcatgacg 2400gcggcgggag ctacatggcc ccgggtgatt tatttttttt gtatctactt ctgacccttt 2460tcaaatatac ggtcaactca tctttcactg gagatgcggc ctgcttggta ttgcgatgtt 2520gtcagcttgg caaattgtgg ctttcgaaaa cacaaaacga ttccttagta gccatgcatt 2580ttaagataac ggaatagaag aaagaggaaa ttaaaaaaaa aaaaaaaaca aacatcccgt 2640tcataacccg tagaatcgcc gctcttcgtg tatcccagta ccagtttaaa cggatctcaa 2700gcttgcatgc aaagatacac atcaatcgca gctggggtac aatcatccat catcccaact 2760ggtacgtcat aacaaaaatc gacaagatgg aaaaagaggt cgcctaaata cagctgcatt 2820ctatgatgcc gggctttgga caagagctct ttctcagctc cgtttgtcct ccctcccttt 2880tcccccttct tgctaaatgc ctttctttac ttctttcttc ccttccctcc cctatcgcag 2940cagcctctcg gtgtaggctt tccacgctgc tgatcggtac cgctctgcct cctctacggg 3000gtctgaggcc ttgaggatgc cccggcccac aatggcaatg tcgctgccgg cgatgccaat 3060cagcttgtgc ggcgtgttgt actgctggcc ctggccgtct ccaccgaccg atccgttggt 3120ctgctggtcc tcgtcttcgg ggggcagctg gcagccgggc gtcatgtgga taaaggcatc 3180gtcgggctcg gtgttgagcg tctcctgcga gatgaagccc atgacaaagt ccttgtgctc 3240ccgggcggcc tcgacgcagg cctgcgtgta ctccttgttc atgaagttgc cctggctgga 3300catttgggcg aggatcagga ggcctcggct cagcggcgcc tcctcgatgc ccgggaagag 3360cgactcgtcg ccctcggcga tggcctttgt taaccggggc gaggagacgg actcgtactg 3420ctgggtgacg gtggtgatgg agacgatgct gcccttgcgg ccgtcgccgg accggttcga 3480gtagatgggc ttgtccagga cgccaatgga gcccatgccg ttgacggcgc cggcgggctc 3540ggcgtccctg gagtcggcgt cgtcgtcaaa cgagtccatg gtgggcgtgc cgacggtgac 3600ggacgtcttg acctcgcagg ggtagcgctc gagccagcgc ttggcgccct gggccagcga 3660ggccaccgac gccttgccgg gcaccatgtt gacgttgaca atgtgcgccc agtcgatgat 3720gcgcgccgac ccgcccgtgt actgcagctc gacggtgtgg ccaatgtcgc caaacttgcg 3780gtcctcgaag atgaggaagc cgtgcttgcg cgccagcgac gccagctggg ctcccgtgcc 3840cgtctccggg tggaagtccc agcccgagac catgtcgtag tgcgtcttga gcacgacaat 3900cgacgggcca atcttgtcgg ccaggtacag cagctcgcgc gctgtcggca cgtcggcgct 3960caggcacagg ttggacgcct tgaggtccat gagcttgaac aggtaagccg tcagcgggtg 4020cgtcgccgtc tcgctcctgg ccgcgaaggt ggccttgagc gtcgggtgtg gtgccatggc 4080tgatgaggct gagagaggct gaggctgcgg ctggttggat agtttaaccc ttagggtgcc 4140gttgtggcgg tttagagggg gggaaaaaaa agagagagat ggcacaattc tgctgtgcga 4200atgacgttgg aagcgcgaca gccgtgcggg aggaagagga gtaggaactg tcggcgattg 4260ggagaatttc gtgcgatccg agtcgtctcg aggcgaggga gttgctttaa tgtcgggctc 4320gtcccctggt caaaattcta gggagcagcg ctggcaacga gagcagagca gcagtagtcg 4380atgctagaaa tcgatagatc cacgatgcca aaaagcttgt tcatttcggc tagcccgtga 4440tcctggcgct tctagggctg aaactgtgtt gttaatgtat tattggctgt gtaactgact 4500tgaatgggga atgaggagcg cgatggattc gcttgcatgt cccctggcca agacgagccg 4560ctttggcggt ttgtgattcg aaggtgtgtc agcggaggcg ccagggcaac acgcactgag 4620ccagccaaca tgcattgctg ccgacatgaa tagacacgcg ccgagcagac ataggagacg 4680tgttgactgt aaaaattcta ctgaatatta gcacgcatgg tctcaataag agcaatagga 4740atgcttgcca atcataagta cgtatgtgct ttttcctgca aatggtacgt acggacagtt 4800catgttgtct gtcatccccc actcaggctc tcatgatcat tttatgggac tggggttttg 4860ctgactgaat ggattcagcc gcacgaaaca aattgggggc catgcagaag ggaagccccc 4920ccagccccct gttcataatt tgttaagagt cggagagctg cctagtatga agcagcaatt 4980gataacgttg actttgcgca tgagctctga agccgggcat atgtatcacg tttctgccta 5040gagccgcacg ggacccaaga agctcttgtc ataaggtatt tatgagtgtt cagctgccaa 5100cgctggttct actttggctc aaccgcatcc cataagctga actttgggag ctgccagaat 5160gtctcttgat gtacagcgat caacaaccgt gcgccggtcg acaactgttc accgatcagg 5220gacgcgaaga ggacccaatc ccggttaacg cacctgctcc gaagaagcaa aagggctatg 5280aggtggtgca gcaaggaatc aaagagctct atccacttga caaggccaat gtcgctcccg 5340atctggagta agtcaaccct gaagtggaag tttgcttctc tgattagtat gtagcatcgt 5400gtttgtccca ggactgggtg caaatcccga agacagctgg aagtccagca agaccgactt 5460caattggacc acgcatacag atggcctcca gagagacttc ccaagagctc ggttgcttct 5520gtatatgtac gactcagcat ggactggcca gctcaaagta aaacaattca tgggcaatat 5580cgcgatgggg ctcttggttg ggctgaggag caagagagag gtaggccaaa cgccagactc 5640gaaccgccag ccaagtctca aactgactgc aggcggccgc catatgcatc ctaggcctat 5700taatattccg gagtatacgt agccggctaa cgttaacaac cggtacctct agaactatag 5760ctagcatgcg caaatttaaa gcgctgatat cgatcgcgcg cagatccata tatagggccc 5820gggttataat tacctcaggt cgacgtccca tggccattcg aattcgtaat catggtcata 5880gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5940cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 6000ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 6060acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 6120gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 6180gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 6240ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 6300cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 6360ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 6420taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6480ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6540ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6600aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6660tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6720agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6780ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 6840tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6900tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6960cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 7020aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 7080atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 7140cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 7200tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 7260atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 7320taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 7380tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 7440gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 7500cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 7560cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 7620gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 7680aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 7740accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 7800ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 7860gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 7920aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 7980taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 8040cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc 8100gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 8160ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 8220cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca 8280taaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 8340attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga 8400gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 8460caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 8520caaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 8580cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 8640agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 8700cacacccgcc gcgcttaatg cgccgctaca gggcgcgtac tatggttgct ttgacgtatg 8760cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 8820ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 8880ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 8940tcacgacgtt gtaaaacgac ggccagtgcc 897032397PRTFusarium oxysporum 32Thr Pro Asp Lys Ala Lys Glu Gln His Pro Lys Leu Glu Thr Tyr Arg1 5 10 15Cys Thr Lys Ala Ser Gly Cys Lys Lys Gln Thr Asn Tyr Ile Val Ala 20 25 30Asp Ala Gly Ile His Gly Ile Arg Gln Lys Asn Gly Ala Gly Cys Gly 35 40 45Asp Trp Gly Gln Lys Pro Asn Ala Thr Ala Cys Pro Asp Glu Ala Ser 50 55 60Cys Ala Lys Asn Cys Ile Leu Ser Gly Met Asp Ser Asn Ala Tyr Lys65 70 75 80Asn Ala Gly Ile Thr Thr Ser Gly Asn Lys Leu Arg Leu Gln Gln Leu 85 90 95Ile Asn Asn Gln Leu Val Ser Pro Arg Val Tyr Leu Leu Glu Glu Asn 100 105 110Lys Lys Lys Tyr Glu Met Leu His Leu Thr Gly Thr Glu Phe Ser Phe 115 120 125Asp Val Glu Met Glu Lys Leu Pro Cys Gly Met Asn Gly Ala Leu Tyr 130 135 140Leu Ser Glu Met Pro Gln Asp Gly Gly Lys Ser Thr Ser Arg Asn Ser145 150 155 160Lys Ala Gly Ala Tyr Tyr Gly Ala Gly Tyr Cys Asp Ala Gln Cys Tyr 165 170 175Val Thr Pro Phe Ile Asn Gly Val Gly Asn Ile Lys Gly Gln Gly Val 180 185 190Cys Cys Asn Glu Leu Asp Ile Trp Glu Ala Asn Ser Arg Ala Thr His 195 200 205Ile Ala Pro His Pro Cys Ser Lys Pro Gly Leu Tyr Gly Cys Thr Gly 210 215 220Asp Glu Cys Gly Ser Ser Gly Ile Cys Asp Lys Ala Gly Cys Gly Trp225 230 235 240Asn His Asn Arg Ile Asn Val Thr Asp Phe Tyr Gly Arg Gly Lys Gln 245 250 255Tyr Lys Val Asp Ser Thr Arg Lys Phe Thr Val Thr Ser Gln Phe Val 260 265 270Ala Asn Lys Gln Gly Asp Leu Ile Glu Leu His Arg His Tyr Ile Gln 275 280 285Asp Asn Lys Val Ile Glu Ser Ala Val Val Asn Ile Ser Gly Pro Pro 290 295 300Lys Ile Asn Phe Ile Asn Asp Lys Tyr Cys Ala Ala Thr Gly Ala Asn305

310 315 320Glu Tyr Met Arg Leu Gly Gly Thr Lys Gln Met Gly Asp Ala Met Ser 325 330 335Arg Gly Met Val Leu Ala Met Ser Val Trp Trp Ser Glu Gly Asp Phe 340 345 350Met Ala Trp Leu Asp Gln Gly Val Ala Gly Pro Cys Asp Ala Thr Glu 355 360 365Gly Asp Pro Lys Asn Ile Val Lys Val Gln Pro Asn Pro Glu Val Thr 370 375 380Phe Ser Asn Ile Arg Ile Gly Glu Ile Gly Ser Thr Ser385 390 39533401PRTHumicola insolens 33Lys Pro Gly Glu Thr Lys Glu Val His Pro Gln Leu Thr Thr Phe Arg1 5 10 15Cys Thr Lys Arg Gly Gly Cys Lys Pro Ala Thr Asn Phe Ile Val Leu 20 25 30Asp Ser Leu Trp His Trp Ile His Arg Ala Glu Gly Leu Gly Pro Gly 35 40 45Gly Cys Gly Asp Trp Gly Asn Pro Pro Pro Lys Asp Val Cys Pro Asp 50 55 60Val Glu Ser Cys Ala Lys Asn Cys Ile Met Glu Gly Ile Pro Asp Tyr65 70 75 80Ser Gln Tyr Gly Val Thr Thr Asn Gly Thr Ser Leu Arg Leu Gln His 85 90 95Ile Leu Pro Asp Gly Arg Val Pro Ser Pro Arg Val Tyr Leu Leu Asp 100 105 110Lys Thr Lys Arg Arg Tyr Glu Met Leu His Leu Thr Gly Phe Glu Phe 115 120 125Thr Phe Asp Val Asp Ala Thr Lys Leu Pro Cys Gly Met Asn Ser Ala 130 135 140Leu Tyr Leu Ser Glu Met His Pro Thr Gly Ala Lys Ser Lys Tyr Asn145 150 155 160Pro Gly Gly Ala Tyr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln Cys Phe 165 170 175Val Thr Pro Phe Ile Asn Gly Leu Gly Asn Ile Glu Gly Lys Gly Ser 180 185 190Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ser Arg Ala Ser His 195 200 205Val Ala Pro His Thr Cys Asn Lys Lys Gly Leu Tyr Leu Cys Glu Gly 210 215 220Glu Glu Cys Ala Phe Glu Gly Val Cys Asp Lys Asn Gly Cys Gly Trp225 230 235 240Asn Asn Tyr Arg Val Asn Val Thr Asp Tyr Tyr Gly Arg Gly Glu Glu 245 250 255Phe Lys Val Asn Thr Leu Lys Pro Phe Thr Val Val Thr Gln Phe Leu 260 265 270Ala Asn Arg Arg Gly Lys Leu Glu Lys Ile His Arg Phe Tyr Val Gln 275 280 285Asp Gly Lys Val Ile Glu Ser Phe Tyr Thr Asn Lys Glu Gly Val Pro 290 295 300Tyr Thr Asn Met Ile Asp Asp Glu Phe Cys Glu Ala Thr Gly Ser Arg305 310 315 320Lys Tyr Met Glu Leu Gly Ala Thr Gln Gly Met Gly Glu Ala Leu Thr 325 330 335Arg Gly Met Val Leu Ala Met Ser Ile Trp Trp Asp Gln Gly Gly Asn 340 345 350Met Glu Trp Leu Asp His Gly Glu Ala Gly Pro Cys Ala Lys Gly Glu 355 360 365Gly Ala Pro Ser Asn Ile Val Gln Val Glu Pro Phe Pro Glu Val Thr 370 375 380Tyr Thr Asn Leu Arg Trp Gly Glu Ile Gly Ser Thr Tyr Gln Glu Leu385 390 395 400Gln34433PRTHypocrea jecorina 34Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp Gln1 5 10 15Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val Val 20 25 30Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr Asn 35 40 45Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn Glu 50 55 60Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser Thr65 70 75 80Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Asp Phe Val Thr 85 90 95Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala Ser 100 105 110Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser Phe 115 120 125Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135 140Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr Asn145 150 155 160Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 165 170 175Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Glu 180 185 190Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser Cys 195 200 205Cys Ser Gln Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala Leu 210 215 220Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly Asp225 230 235 240Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys Asp 245 250 255Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser Phe 260 265 270Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu Thr 275 280 285Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr Val 290 295 300Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser Tyr305 310 315 320Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala Glu 325 330 335Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe Lys 340 345 350Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp Asp Asp 355 360 365Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn Glu 370 375 380Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser Ser385 390 395 400Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val Thr 405 410 415Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro Ser 420 425 430Gly35370PRTHypocrea jecorina 35Gln Pro Gly Thr Ser Thr Pro Glu Val His Pro Lys Leu Thr Thr Tyr1 5 10 15Lys Cys Thr Lys Ser Gly Gly Cys Val Ala Gln Asp Thr Ser Val Val 20 25 30Leu Asp Trp Asn Tyr Arg Trp Met His Asp Ala Asn Tyr Asn Ser Cys 35 40 45Thr Val Asn Gly Gly Val Asn Thr Thr Leu Cys Pro Asp Glu Ala Thr 50 55 60Cys Gly Lys Asn Cys Phe Ile Glu Gly Val Asp Tyr Ala Ala Ser Gly65 70 75 80Val Thr Thr Ser Gly Ser Ser Leu Thr Met Asn Gln Tyr Met Pro Ser 85 90 95Ser Ser Gly Gly Tyr Ser Ser Val Ser Pro Arg Leu Tyr Leu Leu Asp 100 105 110Ser Asp Gly Glu Tyr Val Met Leu Lys Leu Asn Gly Gln Glu Leu Ser 115 120 125Phe Asp Val Asp Leu Ser Ala Leu Pro Cys Gly Glu Asn Gly Ser Leu 130 135 140Tyr Leu Ser Gln Met Asp Glu Asn Gly Gly Ala Asn Gln Tyr Asn Thr145 150 155 160Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cys Asp Ala Gln Cys Pro Val 165 170 175Gln Thr Trp Arg Asn Gly Thr Leu Asn Thr Ser His Gln Gly Phe Cys 180 185 190Cys Asn Glu Met Asp Ile Leu Glu Gly Asn Ser Arg Ala Asn Ala Leu 195 200 205Thr Pro His Ser Cys Thr Ala Thr Ala Cys Asp Ser Ala Gly Cys Gly 210 215 220Phe Asn Pro Tyr Gly Ser Gly Tyr Lys Ser Tyr Tyr Gly Pro Gly Asp225 230 235 240Thr Val Asp Thr Ser Lys Thr Phe Thr Ile Ile Thr Gln Phe Asn Thr 245 250 255Asp Asn Gly Ser Pro Ser Gly Asn Leu Val Ser Ile Thr Arg Lys Tyr 260 265 270Gln Gln Asn Gly Val Asp Ile Pro Ser Ala Gln Pro Gly Gly Asp Thr 275 280 285Ile Ser Ser Cys Pro Ser Ala Ser Ala Tyr Gly Gly Leu Ala Thr Met 290 295 300Gly Lys Ala Leu Ser Ser Gly Met Val Leu Val Phe Ser Ile Trp Asn305 310 315 320Asp Asn Ser Gln Tyr Met Asn Trp Leu Asp Ser Gly Asn Ala Gly Pro 325 330 335Cys Ser Ser Thr Glu Gly Asn Pro Ser Asn Ile Leu Ala Asn Asn Pro 340 345 350Asn Thr His Val Val Phe Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser 355 360 365Thr Thr 37036504PRTPhanerochaete chrysosporium 36Met Arg Thr Ala Leu Ala Leu Ile Leu Ala Leu Ala Ala Phe Ser Ala1 5 10 15Val Ser Ala Gln Gln Ala Gly Thr Ile Thr Ala Glu Thr His Pro Thr 20 25 30Leu Thr Ile Gln Gln Cys Thr Gln Ser Gly Gly Cys Ala Pro Leu Thr 35 40 45 Thr Lys Val Val Leu Asp Val Asn Trp Arg Trp Ile His Ser Thr Thr 50 55 60Gly Tyr Thr Asn Cys Tyr Ser Gly Asn Thr Trp Asp Ala Ile Leu Cys65 70 75 80Pro Asp Pro Val Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp 85 90 95Tyr Thr Gly Thr Phe Gly Ile Leu Pro Ser Gly Thr Ser Val Thr Leu 100 105 110Arg Pro Val Asp Gly Leu Gly Leu Arg Leu Phe Leu Leu Ala Asp Asp 115 120 125Ser His Tyr Gln Met Phe Gln Leu Leu Asn Lys Glu Phe Thr Phe Asp 130 135 140Val Glu Met Pro Asn Met Arg Cys Gly Ser Ser Gly Ala Ile His Leu145 150 155 160Thr Ala Met Asp Ala Asp Gly Gly Leu Ala Lys Tyr Pro Gly Asn Gln 165 170 175Ala Gly Ala Lys Tyr Gly Thr Gly Phe Cys Ser Ala Gln Cys Pro Lys 180 185 190Gly Val Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Leu Gly 195 200 205Thr Thr Ala Thr Thr Gly Thr Gly Phe Phe Gly Ser Cys Cys Thr Asp 210 215 220Ile Ala Leu Trp Glu Ala Asn Asp Asn Ser Ala Ser Phe Ala Pro His225 230 235 240Pro Cys Thr Thr Asn Ser Gln Thr Arg Cys Ser Gly Ser Asp Cys Thr 245 250 255Ala Asp Ser Gly Leu Cys Asp Ala Asp Gly Cys Asn Phe Asn Ser Phe 260 265 270Arg Met Gly Asn Thr Thr Phe Phe Gly Ala Gly Met Ser Val Asp Thr 275 280 285Thr Lys Leu Phe Thr Val Val Thr Gln Phe Ile Thr Ser Asp Asn Thr 290 295 300Ser Met Gly Ala Leu Val Glu Ile His Arg Leu Tyr Ile Gln Asn Gly305 310 315 320Gln Val Ile Gln Asn Ser Val Val Asn Ile Pro Gly Ile Asn Pro Ala 325 330 335Thr Ser Ile Thr Asp Asp Leu Cys Ala Gln Glu Asn Ala Ala Phe Gly 340 345 350Gly Thr Ser Ser Phe Ala Gln His Gly Gly Leu Ala Gln Val Gly Glu 355 360 365Ala Leu Arg Ser Gly Met Val Leu Ala Leu Ser Ile Val Asn Ser Ala 370 375 380Ala Asp Thr Leu Trp Leu Asp Ser Asn Tyr Pro Ala Asp Ala Asp Pro385 390 395 400Ser Ala Pro Gly Val Ala Arg Gly Thr Cys Pro Gln Asp Ser Ala Ser 405 410 415Ile Pro Glu Ala Pro Thr Pro Ser Val Val Phe Ser Asn Ile Lys Leu 420 425 430Gly Asp Ile Gly Thr Thr Phe Gly Ala Gly Ser Ala Leu Phe Ser Gly 435 440 445Arg Ser Pro Pro Gly Pro Val Pro Gly Ser Ala Pro Ala Ser Ser Ala 450 455 460Thr Ala Thr Ala Pro Pro Phe Gly Ser Gln Cys Gly Gly Leu Gly Tyr465 470 475 480Ala Gly Pro Thr Gly Val Cys Pro Ser Pro Tyr Thr Cys Gln Ala Leu 485 490 495Asn Ile Tyr Tyr Ser Gln Cys Ile 50037451PRTPhanerochaete chrysosporium 37Met Arg Thr Ala Leu Ala Leu Ile Leu Ala Leu Ala Ala Phe Ser Ala1 5 10 15Val Ser Ala Gln Gln Ala Gly Thr Ile Thr Ala Glu Thr His Pro Thr 20 25 30Leu Thr Ile Gln Gln Cys Thr Gln Ser Gly Gly Cys Ala Pro Leu Thr 35 40 45 Thr Lys Val Val Leu Asp Val Asn Trp Arg Trp Ile His Ser Thr Thr 50 55 60Gly Tyr Thr Asn Cys Tyr Ser Gly Asn Thr Trp Asp Ala Ile Leu Cys65 70 75 80Pro Asp Pro Val Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp 85 90 95Tyr Thr Gly Thr Phe Gly Ile Leu Pro Ser Gly Thr Ser Val Thr Leu 100 105 110Arg Pro Val Asp Gly Leu Gly Leu Arg Leu Phe Leu Leu Ala Asp Asp 115 120 125Ser His Tyr Gln Met Phe Gln Leu Leu Asn Lys Glu Phe Thr Phe Asp 130 135 140Val Glu Met Pro Asn Met Arg Cys Gly Ser Ser Gly Ala Ile His Leu145 150 155 160Thr Ala Met Asp Ala Asp Gly Gly Leu Ala Lys Tyr Pro Gly Asn Gln 165 170 175Ala Gly Ala Lys Tyr Gly Thr Gly Phe Cys Ser Ala Gln Cys Pro Lys 180 185 190Gly Val Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Leu Gly 195 200 205Thr Thr Ala Thr Thr Gly Thr Gly Phe Phe Gly Ser Cys Cys Thr Asp 210 215 220Ile Ala Leu Trp Glu Ala Asn Asp Asn Ser Ala Ser Phe Ala Pro His225 230 235 240Pro Cys Thr Thr Asn Ser Gln Thr Arg Cys Ser Gly Ser Asp Cys Thr 245 250 255Ala Asp Ser Gly Leu Cys Asp Ala Asp Gly Cys Asn Phe Asn Ser Phe 260 265 270Arg Met Gly Asn Thr Thr Phe Phe Gly Ala Gly Met Ser Val Asp Thr 275 280 285Thr Lys Leu Phe Thr Val Val Thr Gln Phe Ile Thr Ser Asp Asn Thr 290 295 300Ser Met Gly Ala Leu Val Glu Ile His Arg Leu Tyr Ile Gln Asn Gly305 310 315 320Gln Val Ile Gln Asn Ser Val Val Asn Ile Pro Gly Ile Asn Pro Ala 325 330 335Thr Ser Ile Thr Asp Asp Leu Cys Ala Gln Glu Asn Ala Ala Phe Gly 340 345 350Gly Thr Ser Ser Phe Ala Gln His Gly Gly Leu Ala Gln Val Gly Glu 355 360 365Ala Leu Arg Ser Gly Met Val Leu Ala Leu Ser Ile Val Asn Ser Ala 370 375 380Ala Asp Thr Leu Trp Leu Asp Ser Asn Tyr Pro Ala Asp Ala Asp Pro385 390 395 400Ser Ala Pro Gly Val Ala Arg Gly Thr Cys Pro Gln Asp Ser Ala Ser 405 410 415Ile Pro Glu Ala Pro Thr Pro Ser Val Val Phe Ser Asn Ile Lys Leu 420 425 430Gly Asp Ile Gly Thr Thr Phe Gly Ala Gly Ser Ala Leu Phe Pro Ser 435 440 445Gly Arg Ser 45038449PRTPhanerochaete chrysosporium 38Met Val Asp Ile Gln Ile Ala Thr Phe Leu Leu Leu Gly Val Val Gly1 5 10 15Val Ala Ala Gln Gln Val Gly Thr Tyr Ile Pro Glu Asn His Pro Leu 20 25 30Leu Ala Thr Gln Ser Cys Thr Ala Ser Gly Gly Cys Thr Thr Ser Ser 35 40 45Ser Lys Ile Val Leu Asp Ala Asn Arg Arg Trp Ile His Ser Thr Leu 50 55 60Gly Thr Thr Ser Cys Leu Thr Ala Asn Gly Trp Asp Pro Thr Leu Cys65 70 75 80Pro Asp Gly Ile Thr Cys Ala Asn Tyr Cys Ala Leu Asp Gly Val Ser 85 90 95Tyr Ser Ser Thr Tyr Gly Ile Thr Thr Ser Gly Ser Ala Leu Arg Leu 100 105 110Gln Phe Val Thr Gly Thr Asn Ile Gly Ser Arg Val Phe Leu Met Ala 115 120 125Asp Asp Thr His Tyr Arg Thr Phe Gln Leu Leu Asn Gln Glu Leu Ala 130 135 140Phe Asp Val Asp Val Ser Lys Leu Pro Cys Gly Leu Asn Gly Ala Leu145 150 155 160Tyr Phe Val Ala Met Asp Ala Asp Gly Gly Lys Ser Lys Tyr Pro Gly 165 170 175Asn Arg Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys 180 185 190Pro Arg Asp Val Gln Phe Ile Asn Gly Gln Ala Asn Val Gln Gly Trp 195 200 205Asn Ala Thr Ser Ala Thr Thr Gly Thr

Gly Ser Tyr Gly Ser Cys Cys 210 215 220Thr Glu Leu Asp Ile Trp Glu Ala Asn Ser Asn Ala Ala Ala Leu Thr225 230 235 240Pro His Thr Cys Thr Asn Asn Ala Gln Thr Arg Cys Ser Gly Ser Asn 245 250 255Cys Thr Ser Asn Thr Gly Phe Cys Asp Ala Asp Gly Cys Asp Phe Asn 260 265 270Ser Phe Arg Leu Gly Asn Thr Thr Phe Leu Gly Ala Gly Met Ser Val 275 280 285Asp Thr Thr Lys Thr Phe Thr Val Val Thr Gln Phe Ile Thr Ser Asp 290 295 300Asn Thr Ser Thr Gly Asn Leu Thr Glu Ile Arg Arg Phe Tyr Val Gln305 310 315 320Asn Gly Asn Val Ile Pro Asn Ser Val Val Asn Val Thr Gly Ile Gly 325 330 335Ala Val Asn Ser Ile Thr Asp Pro Phe Cys Ser Gln Gln Lys Lys Ala 340 345 350Phe Ile Glu Thr Asn Tyr Phe Ala Gln His Gly Gly Leu Ala Gln Leu 355 360 365Gly Gln Ala Leu Arg Thr Gly Met Val Leu Ala Phe Ser Ile Ser Asp 370 375 380Asp Pro Ala Asn His Met Leu Trp Leu Asp Ser Asn Phe Pro Pro Ser385 390 395 400Ala Asn Pro Ala Val Pro Gly Val Ala Arg Gly Met Cys Ser Ile Thr 405 410 415Ser Gly Asn Pro Ala Asp Val Gly Ile Leu Asn Pro Ser Pro Tyr Val 420 425 430Ser Phe Leu Asn Ile Lys Phe Gly Ser Ile Gly Thr Thr Phe Arg Pro 435 440 445Ala 39516PRTPhanerochaete chrysosporium 39Met Phe Arg Thr Ala Thr Leu Leu Ala Phe Thr Met Ala Ala Met Val1 5 10 15Phe Gly Gln Gln Val Gly Thr Asn Thr Ala Glu Asn His Arg Thr Leu 20 25 30Thr Ser Gln Lys Cys Thr Lys Ser Gly Gly Cys Ser Asn Leu Asn Thr 35 40 45Lys Ile Val Leu Asp Ala Asn Trp Arg Trp Leu His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Gln Trp Asp Ala Thr Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Thr Gly Thr Tyr Gly Ile Thr Ala Ser Gly Ser Ser Leu Lys Leu Gln 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Met Phe Gln Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ala Lys Tyr Pro Thr Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Glu Gly Trp Asn 195 200 205Ala Thr Ser Ala Asn Ala Gly Thr Gly Asn Tyr Gly Thr Cys Cys Thr 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Tyr Thr Pro225 230 235 240His Pro Cys Thr Thr Asn Ala Gln Thr Arg Cys Ser Gly Ser Asp Cys 245 250 255Thr Arg Asp Thr Gly Leu Cys Asp Ala Asp Gly Cys Asp Phe Asn Ser 260 265 270Phe Arg Met Gly Asp Gln Thr Phe Leu Gly Lys Gly Leu Thr Val Asp 275 280 285Thr Ser Lys Pro Phe Thr Val Val Thr Gln Phe Ile Thr Asn Asp Gly 290 295 300Thr Ser Ala Gly Thr Leu Thr Glu Ile Arg Arg Leu Tyr Val Gln Asn305 310 315 320Gly Lys Val Ile Gln Asn Ser Ser Val Lys Ile Pro Gly Ile Asp Pro 325 330 335Val Asn Ser Ile Thr Asp Asn Phe Cys Ser Gln Gln Lys Thr Ala Phe 340 345 350Gly Asp Thr Asn Tyr Phe Ala Gln His Gly Gly Leu Lys Gln Val Gly 355 360 365Glu Ala Leu Arg Thr Gly Met Val Leu Ala Leu Ser Ile Trp Asp Asp 370 375 380Tyr Ala Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr Asn Lys385 390 395 400Asp Pro Ser Thr Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr Ser 405 410 415Gly Val Pro Ala Gln Ile Glu Ala Gln Ser Pro Asn Ala Tyr Val Val 420 425 430Phe Ser Asn Ile Lys Phe Gly Asp Leu Asn Thr Thr Tyr Thr Gly Thr 435 440 445Val Ser Ser Ser Ser Val Ser Ser Ser His Ser Ser Thr Ser Thr Ser 450 455 460Ser Ser His Ser Ser Ser Ser Thr Pro Pro Thr Gln Pro Thr Gly Val465 470 475 480Thr Val Pro Gln Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Ser 485 490 495Thr Thr Cys Ala Ser Pro Tyr Thr Cys His Val Leu Asn Pro Tyr Tyr 500 505 510Ser Gln Cys Tyr 51540516PRTPhanerochaete chrysosporium 40Met Phe Arg Thr Ala Thr Leu Leu Ala Phe Thr Met Ala Ala Met Val1 5 10 15Phe Gly Gln Gln Val Gly Thr Asn Thr Ala Glu Asn His Arg Thr Leu 20 25 30Thr Ser Gln Lys Cys Thr Lys Ser Gly Gly Cys Ser Asn Leu Asn Thr 35 40 45Lys Ile Val Leu Asp Ala Asn Trp Arg Trp Leu His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Gln Trp Asp Ala Thr Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Thr Gly Thr Tyr Gly Ile Thr Ala Ser Gly Ser Ser Leu Lys Leu Gln 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Met Phe Gln Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ala Lys Tyr Pro Thr Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Glu Gly Trp Asn 195 200 205Ala Thr Ser Ala Asn Ala Gly Thr Gly Asn Tyr Gly Thr Cys Cys Thr 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Tyr Thr Pro225 230 235 240His Pro Cys Thr Thr Asn Ala Gln Thr Arg Cys Ser Gly Ser Asp Cys 245 250 255Thr Arg Asp Thr Gly Leu Cys Asp Ala Asp Gly Cys Asp Phe Asn Ser 260 265 270Phe Arg Met Gly Asp Gln Thr Phe Leu Gly Lys Gly Leu Thr Val Asp 275 280 285Thr Ser Lys Pro Phe Thr Val Val Thr Gln Phe Ile Thr Asn Asp Gly 290 295 300Thr Ser Ala Gly Thr Leu Thr Glu Ile Arg Arg Leu Tyr Val Gln Asn305 310 315 320Gly Lys Val Ile Gln Asn Ser Ser Val Lys Ile Pro Gly Ile Asp Leu 325 330 335Val Asn Ser Ile Thr Asp Asn Phe Cys Ser Gln Gln Lys Thr Ala Phe 340 345 350Gly Asp Thr Asn Tyr Phe Ala Gln His Gly Gly Leu Lys Gln Val Gly 355 360 365Glu Ala Leu Arg Thr Gly Met Val Leu Ala Leu Ser Ile Trp Asp Asp 370 375 380Tyr Ala Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr Asn Lys385 390 395 400Asp Pro Ser Thr Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr Ser 405 410 415Gly Val Pro Ala Gln Ile Glu Ala Gln Ser Pro Asn Ala Tyr Val Val 420 425 430Phe Ser Asn Ile Lys Phe Gly Asp Leu Asn Thr Thr Tyr Thr Gly Thr 435 440 445Val Ser Ser Ser Ser Val Ser Ser Ser His Ser Ser Thr Ser Thr Ser 450 455 460Ser Ser His Ser Ser Ser Ser Thr Pro Pro Thr Gln Pro Thr Gly Val465 470 475 480Thr Val Pro Gln Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Ser 485 490 495Thr Thr Cys Ala Ser Pro Tyr Thr Cys His Val Leu Asn Pro Tyr Tyr 500 505 510Ser Gln Cys Tyr 51541516PRTPhanerochaete chrysosporium 41Met Phe Arg Thr Ala Thr Leu Leu Ala Phe Thr Met Ala Ala Met Val1 5 10 15 Phe Gly Gln Gln Val Gly Thr Asn Thr Ala Arg Ser His Pro Ala Leu 20 25 30Thr Ser Gln Lys Cys Thr Lys Ser Gly Gly Cys Ser Asn Leu Asn Thr 35 40 45Lys Ile Val Leu Asp Ala Asn Trp Arg Trp Leu His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Gln Trp Asp Ala Thr Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Thr Gly Thr Tyr Gly Ile Thr Ala Ser Gly Ser Ser Leu Lys Leu Gln 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Met Phe Gln Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ala Lys Tyr Pro Thr Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Glu Gly Trp Asn 195 200 205Ala Thr Ser Ala Asn Ala Gly Thr Gly Asn Tyr Gly Thr Cys Cys Thr 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Tyr Thr Pro225 230 235 240His Pro Cys Thr Thr Asn Ala Gln Thr Arg Cys Ser Gly Ser Asp Cys 245 250 255Thr Arg Asp Thr Gly Leu Cys Asp Ala Asp Gly Cys Asp Phe Asn Ser 260 265 270Phe Arg Met Gly Asp Gln Thr Phe Leu Gly Lys Gly Leu Thr Val Asp 275 280 285Thr Ser Lys Pro Phe Thr Val Val Thr Gln Phe Ile Thr Asn Asp Gly 290 295 300Thr Ser Ala Gly Thr Leu Thr Glu Ile Arg Arg Leu Tyr Val Gln Asn305 310 315 320Gly Lys Val Ile Gln Asn Ser Ser Val Lys Ile Pro Gly Ile Asp Pro 325 330 335Val Asn Ser Ile Thr Asp Asn Phe Cys Ser Gln Gln Lys Thr Ala Phe 340 345 350Gly Asp Thr Asn Tyr Phe Ala Gln His Gly Gly Leu Lys Gln Val Gly 355 360 365Glu Ala Leu Arg Thr Gly Met Val Leu Ala Leu Ser Ile Trp Asp Asp 370 375 380Tyr Ala Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr Asn Lys385 390 395 400Asp Pro Ser Thr Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr Ser 405 410 415Gly Val Pro Ala Gln Ile Glu Ala Gln Ser Pro Asn Ala Tyr Val Val 420 425 430Phe Ser Asn Ile Lys Phe Gly Asp Leu Asn Thr Thr Tyr Thr Gly Thr 435 440 445Val Ser Ser Ser Ser Val Ser Ser Ser His Ser Ser Thr Ser Thr Ser 450 455 460Ser Ser His Ser Ser Ser Ser Thr Pro Pro Thr Gln Pro Thr Gly Val465 470 475 480Thr Val Pro Gln Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Ser 485 490 495Thr Thr Cys Ala Ser Pro Tyr Thr Cys His Val Leu Asn Pro Tyr Tyr 500 505 510Ser Gln Cys Tyr 51542511PRTPhanerochaete chrysosporium 42Met Phe Arg Ala Ala Ala Leu Leu Ala Phe Thr Cys Leu Ala Met Val1 5 10 15Ser Gly Gln Gln Ala Gly Thr Asn Thr Ala Glu Asn His Pro Gln Leu 20 25 30Gln Ser Gln Gln Cys Thr Thr Ser Gly Gly Cys Lys Pro Leu Ser Thr 35 40 45Lys Val Val Leu Asp Ser Asn Trp Arg Trp Val His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asn Thr Ser Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Ser Gly Thr Tyr Gly Ile Thr Ser Thr Gly Thr Ala Leu Thr Leu Lys 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Leu Leu Lys Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ser Lys Tyr Pro Gly Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Lys Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Gly Asn Trp Thr 195 200 205Glu Thr Gly Ser Asn Thr Gly Thr Gly Ser Tyr Gly Thr Cys Cys Ser 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Phe Thr Pro225 230 235 240His Pro Cys Thr Thr Thr Gly Gln Thr Arg Cys Ser Gly Asp Asp Cys 245 250 255Ala Arg Asn Thr Gly Leu Cys Asp His Gly Asp Gly Cys Asp Phe Asn 260 265 270Ser Phe Arg Met Gly Asp Lys Thr Phe Leu Gly Lys Gly Met Thr Val 275 280 285Asp Thr Ser Lys Pro Phe Thr Asp Val Thr Gln Phe Leu Thr Asn Asp 290 295 300Asn Thr Ser Thr Gly Thr Leu Ser Glu Ile Arg Arg Ile Tyr Ile Gln305 310 315 320Asn Gly Lys Val Ile Gln Asn Ser Val Ala Asn Ile Pro Gly Val Asp 325 330 335Pro Val Asn Ser Ile Thr Asp Asn Phe Cys Ala Gln Gln Lys Thr Ala 340 345 350Phe Gly Asp Thr Asn Trp Phe Ala Gln Lys Gly Gly Leu Lys Gln Met 355 360 365Gly Glu Ala Leu Gly Asn Gly Met Val Leu Ala Leu Ser Ile Trp Asp 370 375 380Asp His Ala Ala Asn Met Leu Trp Leu Asp Ser Asp Tyr Pro Thr Asp385 390 395 400Lys Asp Pro Ser Ala Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr 405 410 415Ser Gly Val Pro Ser Asp Val Glu Ser Gln Val Pro Asn Ser Gln Val 420 425 430Val Phe Ser Asn Ile Lys Phe Gly Asp Ile Gly Ser Thr Phe Ser Gly 435 440 445Thr Ser Ser Pro Asn Pro Pro Gly Gly Ser Thr Thr Ser Ser Pro Val 450 455 460Thr Thr Ser Pro Thr Pro Pro Pro Thr Gly Pro Thr Val Pro Gln Trp465 470 475 480Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Ser Thr Thr Cys Ala Ser 485 490 495Pro Tyr Thr Cys His Val Leu Asn Pro Tyr Tyr Ser Gln Cys Tyr 500 505 51043510PRTPhanerochaete chrysosporium 43Met Phe Arg Ala Ala Ala Leu Leu Ala Phe Thr Cys Leu Ala Met Val1 5 10 15Ser Gly Gln Gln Ala Gly Thr Asn Thr Ala Glu Asn His Pro Gln Leu 20 25 30Gln Ser Gln Gln Cys Thr Thr Ser Gly Gly Cys Lys Pro Leu Ser Thr 35 40 45Lys Val Val Leu Asp Ser Asn Trp Arg Trp Val His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Ser Gly Thr Tyr Gly Ile Thr Ser Thr Gly Thr Ala Leu Thr Leu Lys 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Leu Leu Lys Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu

Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ser Lys Tyr Pro Gly Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Lys Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Gly Asn Trp Thr 195 200 205Glu Thr Gly Ser Asn Thr Gly Thr Gly Ser Tyr Gly Thr Cys Cys Ser 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Phe Thr Pro225 230 235 240His Pro Cys Thr Thr Thr Gly Gln Thr Arg Cys Ser Gly Asp Asp Cys 245 250 255Ala Arg Asn Thr Gly Leu Cys Asp Gly Asp Gly Cys Asp Phe Asn Ser 260 265 270Phe Arg Met Gly Asp Lys Thr Phe Leu Gly Lys Gly Met Thr Val Asp 275 280 285Thr Ser Lys Pro Phe Thr Val Val Thr Gln Phe Leu Thr Asn Asp Asn 290 295 300Thr Ser Thr Gly Thr Leu Ser Glu Ile Arg Arg Ile Tyr Ile Gln Asn305 310 315 320Gly Lys Val Ile Gln Asn Ser Val Ala Asn Ile Pro Gly Val Asp Pro 325 330 335Val Asn Ser Ile Thr Asp Asn Phe Cys Ala Gln Gln Lys Thr Ala Phe 340 345 350Gly Asp Thr Asn Trp Phe Ala Gln Lys Gly Gly Leu Lys Gln Met Gly 355 360 365Glu Ala Leu Gly Asn Gly Met Val Leu Ala Leu Ser Ile Trp Asp Asp 370 375 380His Ala Ala Asn Met Leu Trp Leu Asp Ser Asp Tyr Pro Thr Asp Lys385 390 395 400Asp Pro Ser Ala Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr Ser 405 410 415Gly Val Pro Ser Asp Val Glu Ser Gln Val Pro Asn Ser Gln Val Val 420 425 430Phe Ser Asn Ile Lys Phe Gly Asp Ile Gly Ser Thr Phe Ser Gly Thr 435 440 445Ser Ser Pro Asn Pro Pro Gly Gly Ser Thr Thr Ser Ser Pro Val Thr 450 455 460Thr Ser Pro Thr Pro Pro Pro Thr Gly Pro Thr Val Pro Gln Trp Gly465 470 475 480Gln Cys Gly Gly Ile Gly Tyr Ser Gly Ser Thr Thr Cys Ala Ser Pro 485 490 495Tyr Thr Cys His Val Leu Asn Pro Tyr Tyr Ser Gln Cys Tyr 500 505 51044540PRTPhanerochaete chrysosporium 44Met Phe Arg Ala Ala Ala Leu Leu Ala Phe Thr Cys Leu Ala Met Val1 5 10 15Ser Gly Gln Gln Ala Gly Thr Asn Thr Ala Glu Asn His Pro Gln Leu 20 25 30Gln Ser Gln Gln Cys Thr Thr Ser Gly Gly Cys Lys Pro Leu Ser Thr 35 40 45Lys Val Val Leu Asp Ser Asn Trp Arg Trp Val His Ser Thr Ser Gly 50 55 60Tyr Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser Leu Cys Pro65 70 75 80Asp Gly Lys Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Ser Gly Thr Tyr Gly Ile Thr Ser Thr Gly Thr Ala Leu Thr Leu Lys 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Ala Asp 115 120 125Asp Thr His Tyr Gln Leu Leu Lys Leu Leu Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Leu Ser Ala Met Asp Ala Asp Gly Gly Met Ser Lys Tyr Pro Gly Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Lys Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Gly Asn Trp Thr 195 200 205Glu Thr Gly Ser Asn Thr Gly Thr Gly Ser Tyr Gly Thr Cys Cys Ser 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Phe Thr Pro225 230 235 240His Pro Cys Thr Thr Thr Gly Gln Thr Arg Cys Ser Gly Asp Asp Cys 245 250 255Ala Arg Asn Thr Gly Leu Cys Asp Gly Asp Gly Cys Asp Phe Asn Ser 260 265 270Phe Arg Met Gly Asp Lys Thr Phe Leu Gly Lys Gly Met Thr Val Asp 275 280 285Thr Ser Lys Pro Phe Thr Val Val Thr Gln Phe Leu Thr Asn Asp Asn 290 295 300Thr Ser Thr Gly Thr Leu Ser Glu Ile Arg Arg Ile Tyr Ile Gln Asn305 310 315 320Gly Lys Val Ile Gln Asn Ser Val Ala Asn Ile Pro Gly Val Asp Pro 325 330 335Val Asn Ser Ile Thr Asp Asn Phe Cys Ala Gln Gln Lys Thr Ala Phe 340 345 350Gly Asp Thr Asn Trp Phe Ala Gln Lys Gly Gly Leu Lys Gln Met Gly 355 360 365Glu Ala Leu Gly Asn Gly Met Val Leu Ala Leu Ser Ile Trp Asp Asp 370 375 380His Ala Ala Asn Met Leu Trp Leu Asp Ser Asp Tyr Pro Thr Asp Lys385 390 395 400Asp Pro Ser Ala Pro Gly Val Ala Arg Gly Thr Cys Ala Thr Thr Ser 405 410 415Gly Val Pro Ser Asp Val Glu Ser Gln Val Pro Asn Ser Gln Val Val 420 425 430Phe Ser Asn Ile Lys Phe Gly Asp Ile Gly Ser Thr Phe Ser Gly Thr 435 440 445Ser Ser Pro Asn Pro Pro Gly Gly Ser Thr Thr Ser Ser Pro Val Thr 450 455 460Thr Ser Pro Thr Pro Pro Pro Thr Gly Pro Thr Val Pro Gln Trp Gly465 470 475 480Gln Cys Gly Gly Ile Gly Tyr Ser Gly Ser Thr Thr Cys Ala Ser Pro 485 490 495Tyr Thr Cys His Val Leu Asn Pro Cys Glu Ser Ile Leu Ser Leu Gln 500 505 510Arg Ser Ser Asn Ala Asp Gln Tyr Leu Gln Thr Thr Arg Ser Ala Thr 515 520 525Lys Arg Arg Leu Asp Thr Ala Leu Gln Pro Arg Lys 530 535 54045523PRTIrpex lacteus 45Met Phe Arg Lys Ala Ala Leu Leu Ala Phe Ser Phe Leu Ala Ile Ala1 5 10 15His Gly Gln Gln Val Gly Thr Asn Gln Ala Glu Asn His Pro Ser Leu 20 25 30Pro Ser Gln Lys Cys Thr Ala Ser Gly Cys Thr Thr Ser Ser Thr Ser 35 40 45Val Val Leu Asp Ala Asn Trp Arg Trp Val His Thr Thr Thr Gly Tyr 50 55 60Thr Asn Cys Tyr Thr Gly Gln Thr Trp Asp Ala Ser Ile Cys Pro Asp65 70 75 80Gly Val Thr Cys Ala Lys Ala Cys Ala Leu Asp Gly Ala Asp Tyr Ser 85 90 95Gly Thr Tyr Gly Ile Thr Thr Ser Gly Asn Ala Leu Thr Leu Gln Phe 100 105 110Val Lys Gly Thr Asn Val Gly Ser Arg Val Tyr Leu Leu Gln Asp Ala 115 120 125Ser Asn Tyr Gln Met Phe Gln Leu Ile Asn Gln Glu Phe Thr Phe Asp 130 135 140Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Val Tyr Leu145 150 155 160Ser Gln Met Asp Gln Asp Gly Gly Val Ser Arg Phe Pro Thr Asn Thr 165 170 175Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg 180 185 190Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Glu Gly Trp Thr Gly 195 200 205Ser Ser Thr Asp Ser Asn Ser Gly Thr Gly Asn Tyr Gly Thr Cys Cys 210 215 220Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Val Ala Ala Ala Tyr Thr225 230 235 240Pro His Pro Cys Ser Val Asn Gln Gln Thr Arg Cys Thr Gly Ala Asp 245 250 255Cys Gly Gln Gly Asp Asp Arg Tyr Asp Gly Val Cys Asp Pro Asp Gly 260 265 270Cys Asp Phe Asn Ser Phe Arg Met Gly Asp Gln Thr Phe Leu Gly Lys 275 280 285Gly Leu Thr Val Asp Thr Ser Arg Lys Phe Thr Ile Val Thr Gln Phe 290 295 300Ile Ser Asp Asp Gly Thr Thr Ser Gly Asn Leu Ala Glu Ile Arg Arg305 310 315 320Phe Tyr Val Gln Asp Gly Asn Val Ile Pro Asn Ser Lys Val Ser Ile 325 330 335Ala Gly Ile Asp Ala Val Asn Ser Ile Thr Asp Asp Phe Cys Thr Gln 340 345 350Gln Lys Thr Ala Phe Gly Asp Thr Asn Arg Phe Ala Ala Gln Gly Gly 355 360 365Leu Lys Gln Met Gly Ala Ala Leu Lys Ser Gly Met Val Leu Ala Leu 370 375 380Ser Leu Trp Asp Asp His Ala Ala Asn Met Leu Trp Leu Asp Ser Asp385 390 395 400Tyr Pro Thr Thr Ala Asp Ala Ser Asn Pro Gly Val Ala Arg Gly Thr 405 410 415Cys Pro Thr Thr Ser Gly Phe Pro Arg Asp Val Glu Ser Gln Ser Gly 420 425 430Ser Ala Thr Val Thr Tyr Ser Asn Ile Lys Trp Gly Asp Leu Asn Ser 435 440 445Thr Phe Thr Gly Thr Leu Thr Thr Pro Ser Gly Ser Ser Ser Pro Ser 450 455 460Ser Pro Ala Ser Thr Ser Gly Ser Ser Thr Ser Ala Ser Ser Ser Ala465 470 475 480Ser Val Pro Thr Gln Ser Gly Thr Val Ala Gln Trp Ala Gln Cys Gly 485 490 495Gly Ile Gly Tyr Ser Gly Ala Thr Thr Cys Val Ser Pro Tyr Thr Cys 500 505 510His Val Val Asn Ala Tyr Tyr Ser Gln Cys Tyr 515 52046526PRTIrpex lacteus 46Met Phe His Lys Ala Val Leu Val Ala Phe Ser Leu Val Thr Ile Val1 5 10 15His Gly Gln Gln Ala Gly Thr Gln Thr Ala Glu Asn His Pro Gln Leu 20 25 30Ser Ser Gln Lys Cys Thr Ala Gly Gly Ser Cys Thr Ser Ala Ser Thr 35 40 45Ser Val Val Leu Asp Ser Asn Trp Arg Trp Val His Thr Thr Ser Gly 50 55 60 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Ala Ser Ile Cys Ser65 70 75 80Asp Pro Val Ser Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95Ala Gly Thr Tyr Gly Ile Thr Thr Ser Gly Asp Ala Leu Thr Leu Lys 100 105 110Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr Leu Met Glu Asp 115 120 125Glu Thr Asn Tyr Gln Met Phe Lys Leu Met Asn Gln Glu Phe Thr Phe 130 135 140Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Val Tyr145 150 155 160Phe Val Gln Met Asp Gln Asp Gly Gly Thr Ser Lys Phe Pro Asn Asn 165 170 175Lys Ala Gly Ala Lys Phe Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Gln Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile Val Asp Trp Thr 195 200 205Ala Ser Ala Gly Asp Ala Asn Ser Gly Thr Gly Ser Phe Gly Thr Cys 210 215 220Cys Gln Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Ala Ala Tyr225 230 235 240Thr Pro His Pro Cys Thr Val Thr Glu Gln Thr Arg Cys Ser Gly Ser 245 250 255Asp Cys Gly Gln Gly Ser Asp Arg Phe Asn Gly Ile Cys Asp Pro Asp 260 265 270Gly Cys Asp Phe Asn Ser Phe Arg Met Gly Asn Thr Glu Phe Tyr Gly 275 280 285Lys Gly Leu Thr Val Asp Thr Ser Gln Lys Phe Thr Ile Val Thr Gln 290 295 300Phe Ile Ser Asp Asp Gly Thr Ala Asp Gly Asn Leu Ala Glu Ile Arg305 310 315 320Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Asn Ser Val Val Gln 325 330 335Ile Thr Gly Ile Asp Pro Val Asn Ser Ile Thr Glu Asp Phe Cys Thr 340 345 350Gln Gln Lys Thr Val Phe Gly Asp Thr Asn Asn Phe Ala Ala Lys Gly 355 360 365Gly Leu Lys Gln Met Gly Glu Ala Val Lys Asn Gly Met Val Leu Ala 370 375 380Leu Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser385 390 395 400Asp Tyr Pro Thr Thr Ala Asp Pro Ser Gln Pro Gly Val Ala Arg Gly 405 410 415Thr Cys Pro Thr Thr Ser Gly Val Pro Ser Gln Val Glu Gly Gln Glu 420 425 430Gly Ser Ser Ser Val Ile Tyr Ser Asn Ile Lys Phe Gly Asp Leu Asn 435 440 445Ser Thr Phe Thr Gly Thr Leu Thr Asn Pro Ser Ser Pro Ala Gly Pro 450 455 460Pro Val Thr Ser Ser Pro Ser Glu Pro Ser Gln Ser Thr Gln Pro Ser465 470 475 480Gln Pro Ala Gln Pro Thr Gln Pro Ala Gly Thr Ala Ala Gln Trp Ala 485 490 495Gln Cys Gly Gly Met Gly Phe Thr Gly Pro Thr Val Cys Ala Ser Pro 500 505 510Phe Thr Cys His Val Leu Asn Pro Tyr Tyr Ser Gln Cys Tyr 515 520 52547517PRTIrpex lacteus 47Met Phe Pro Lys Ala Ser Leu Ile Ala Leu Ser Phe Ile Ala Ala Val1 5 10 15Tyr Gly Gln Gln Val Gly Thr Gln Met Ala Glu Val His Pro Lys Leu 20 25 30Pro Ser Gln Leu Cys Thr Lys Ser Gly Cys Thr Asn Gln Asn Thr Ala 35 40 45Val Val Leu Asp Ala Asn Trp Arg Trp Leu His Thr Thr Ser Gly Tyr 50 55 60Thr Asn Cys Tyr Thr Gly Asn Ser Trp Asp Ala Thr Leu Cys Pro Asp65 70 75 80Ala Thr Thr Cys Ala Gln Asn Cys Ala Val Asp Gly Ala Asp Tyr Ser 85 90 95Gly Thr Tyr Gly Ile Thr Thr Ser Gly Asn Ala Leu Thr Leu Lys Phe 100 105 110Lys Thr Gly Thr Asn Val Gly Ser Arg Val Tyr Leu Met Gln Thr Asp 115 120 125Thr Ala Tyr Gln Met Phe Gln Leu Leu Asn Gln Glu Phe Thr Phe Asp 130 135 140Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Leu145 150 155 160Ser Gln Met Asp Gln Asp Gly Gly Leu Ser Lys Phe Pro Thr Asn Lys 165 170 175Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro His 180 185 190Asp Ile Lys Phe Ile Asn Gly Met Ala Asn Val Ala Gly Trp Ala Gly 195 200 205Ser Ala Ser Asp Pro Asn Ala Gly Ser Gly Thr Leu Gly Thr Cys Cys 210 215 220Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Asp Ala Ala Ala Phe Thr225 230 235 240Pro His Pro Cys Ser Val Asp Gly Gln Thr Gln Cys Ser Gly Thr Gln 245 250 255Cys Gly Asp Asp Asp Glu Arg Tyr Ser Gly Leu Cys Asp Lys Asp Gly 260 265 270Cys Asp Phe Asn Ser Phe Arg Met Gly Asp Lys Ser Phe Leu Gly Lys 275 280 285Gly Met Thr Val Asp Thr Ser Arg Lys Phe Thr Val Val Thr Gln Phe 290 295 300Val Thr Thr Asp Gly Thr Thr Asn Gly Asp Leu His Glu Ile Arg Arg305 310 315 320Leu Tyr Val Gln Asp Gly Lys Val Ile Gln Asn Ser Val Val Ser Ile 325 330 335Pro Gly Ile Asp Ala Val Asp Ser Ile Thr Asp Asn Phe Cys Ala Gln 340 345 350Gln Lys Ser Val Phe Gly Asp Thr Asn Tyr Phe Ala Thr Leu Gly Gly 355 360 365Leu Lys Lys Met Gly Ala Ala Leu Lys Ser Gly Met Val Leu Ala Met 370 375 380Ser Val Trp Asp Asp His Ala Ala Ser Met Gln Trp Leu Asp Ser Asn385 390 395 400Tyr Pro Ala Asp Gly Asp Ala Thr Lys Pro Gly Val Ala Arg Gly Thr 405 410 415Cys Ser Ala Asp Ser Gly Leu Pro Thr Asn Val Glu Ser Gln Ser Ala 420 425 430Ser Ala Ser Val Thr Phe Ser Asn Ile Lys Trp Gly Asp Ile Asn Thr 435 440 445Thr Phe Thr Gly Thr Gly Ser Thr Ser Pro Ser Ser Pro Ala Gly Pro 450 455 460Val Ser Ser Ser Thr Ser Val Ala Ser Gln Pro Thr Gln Pro Ala Gln465 470 475 480Gly Thr Val Ala Gln Trp Gly Gln Cys Gly Gly Thr Gly Phe Thr Gly 485 490 495Pro Thr Val Cys Ala Ser Pro Phe Thr Cys His Val Val Asn Pro Tyr 500

505 510Tyr Ser Gln Cys Tyr 51548423PRTAlternaria alternata 48Met Thr Trp Gln Ser Cys Thr Ala Lys Gly Ser Cys Thr Asn Lys Asn1 5 10 15Gly Lys Ile Val Ile Asp Ala Asn Trp Arg Trp Leu His Lys Lys Glu 20 25 30Gly Tyr Asp Asn Cys Tyr Thr Gly Asn Glu Trp Asp Ala Thr Ala Cys 35 40 45Pro Asp Asn Lys Ala Cys Ala Ala Asn Cys Ala Val Asp Gly Ala Asp 50 55 60Tyr Ser Gly Thr Tyr Gly Ile Thr Ala Gly Ser Asn Ser Leu Lys Leu65 70 75 80Lys Phe Ile Thr Lys Gly Ser Tyr Ser Thr Asn Ile Gly Ser Arg Thr 85 90 95Tyr Leu Met Lys Asp Asp Thr Thr Tyr Glu Met Phe Lys Phe Thr Gly 100 105 110Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys Gly 115 120 125Phe Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu 130 135 140Lys Lys Tyr Ser Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr145 150 155 160Cys Asp Ala Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Gly 165 170 175Asn Val Glu Gly Trp Lys Pro Ser Ser Asn Asp Ala Asn Ala Gly Val 180 185 190Gly Gly His Gly Ser Cys Cys Ala Glu Met Asp Ile Trp Glu Ala Asn 195 200 205Ser Val Ser Thr Ala Val Thr Pro His Ser Cys Ser Thr Ile Glu Gln 210 215 220Ser Arg Cys Asp Gly Asp Gly Cys Gly Gly Thr Tyr Ser Ala Asp Arg225 230 235 240Tyr Ala Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg 245 250 255Met Gly Val Lys Asp Phe Tyr Gly Lys Gly Lys Thr Val Asp Thr Ser 260 265 270Lys Lys Phe Thr Val Val Thr Gln Phe Ile Gly Thr Gly Asp Ala Met 275 280 285Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Thr Ile Ala Gln Pro 290 295 300Ala Ser Ala Val Pro Gly Val Glu Gly Asn Ser Ile Thr Thr Lys Phe305 310 315 320Cys Asp Gln Gln Lys Ala Val Phe Gly Asp Thr Tyr Thr Phe Lys Asp 325 330 335Lys Gly Gly Met Ala Asn Met Ala Lys Ala Leu Ala Asn Gly Met Val 340 345 350Leu Val Met Ser Leu Trp Asp Asp His Tyr Ser Asn Met Leu Trp Leu 355 360 365Asp Ser Thr Tyr Pro Thr Asp Lys Asn Pro Asp Thr Asp Leu Gly Thr 370 375 380Gly Arg Gly Glu Cys Glu Thr Ser Ser Gly Val Pro Ala Asp Val Glu385 390 395 400Ser Gln His Ala Asp Ala Thr Val Val Tyr Ser Asn Ile Lys Phe Gly 405 410 415Pro Leu Asn Ser Thr Phe Gly 42049438PRTLeptosphaeria maculans 49Met Tyr Arg Ser Leu Ile Phe Ala Thr Ser Leu Leu Ser Leu Ala Lys1 5 10 15Gly Gln Leu Val Gly Asn Leu Tyr Cys Lys Gly Ser Cys Thr Ala Lys 20 25 30Asn Gly Lys Val Val Ile Asp Ala Asn Trp Arg Trp Leu His Val Lys 35 40 45Gly Gly Tyr Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asn Ala Thr Ala 50 55 60Cys Pro Asp Asn Lys Ser Cys Ala Thr Asn Cys Ala Ile Asp Gly Ala65 70 75 80Asp Tyr Arg Arg Leu Arg His Tyr Cys Glu Arg Gln Leu Leu Gly Thr 85 90 95Glu Val His His Gln Gly Leu Tyr Ser Thr Asn Ile Gly Ser Arg Thr 100 105 110Tyr Leu Met Gln Asp Asp Ser Thr Tyr Gln Leu Phe Lys Phe Thr Gly 115 120 125Ser Gln Glu Phe Thr Phe Asp Val Asp Leu Ser Asn Leu Pro Cys Gly 130 135 140Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu145 150 155 160Lys Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr 165 170 175Cys Asp Ala Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Gly 180 185 190Asn Val Glu Gly Trp Gln Pro Ser Lys Asn Asp Gln Asn Ala Gly Val 195 200 205Gly Gly His Gly Ser Cys Cys Ala Glu Met Asp Ile Trp Glu Ala Asn 210 215 220Ser Val Ser Thr Ala Val Thr Pro His Ser Cys Ser Thr Ile Glu Gln225 230 235 240Ser Arg Cys Asp Gly Asp Gly Cys Gly Gly Thr Tyr Ser Ala Asp Arg 245 250 255Tyr Ala Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg 260 265 270Met Gly Val Lys Asp Phe Tyr Gly Lys Gly Lys Thr Val Asp Thr Ser 275 280 285Lys Lys Phe Thr Val Val Thr Gln Phe Ile Gly Ser Gly Asp Ala Met 290 295 300Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Thr Ile Pro Gln Pro305 310 315 320Asp Ser Thr Ile Pro Gly Val Thr Gly Asn Ser Ile Thr Thr Phe Phe 325 330 335Cys Asp Ala Gln Lys Lys Ala Phe Gly Asp Lys Tyr Thr Phe Lys Asp 340 345 350Lys Gly Gly Met Ala Asn Met Pro Ser Thr Cys Asn Gly Met Val Leu 355 360 365Val Met Ser Leu Trp Asp Asp His Tyr Ser Asn Met Leu Trp Leu Asp 370 375 380Ser Thr Tyr Pro Thr Asp Lys Asn Pro Asp Thr Asp Ala Gly Ser Gly385 390 395 400Arg Gly Glu Cys Ala Ile Thr Ser Gly Val Pro Ala Asp Val Glu Ser 405 410 415Gln His Pro Asp Ala Ser Val Ile Tyr Ser Asn Ile Lys Phe Gly Pro 420 425 430Ile Asn Thr Thr Phe Gly 43550452PRTCryphonectria parasitica 50Met Phe Ser Lys Phe Ala Leu Thr Gly Ser Leu Leu Ala Gly Ala Val1 5 10 15Asn Ala Gln Gly Val Gly Thr Gln Gln Thr Glu Thr His Pro Gln Met 20 25 30Thr Trp Gln Ser Cys Thr Ser Pro Ser Ser Cys Thr Thr Asn Gln Gly 35 40 45Glu Val Val Ile Asp Ser Asn Trp Arg Trp Val His Asp Lys Asp Gly 50 55 60Tyr Val Asn Cys Tyr Thr Gly Asn Thr Trp Asn Thr Thr Leu Cys Pro65 70 75 80Asp Asp Lys Thr Cys Ala Ala Asn Cys Val Leu Asp Gly Ala Asp Tyr 85 90 95Ser Ser Thr Tyr Gly Ile Thr Thr Ser Gly Asn Ala Leu Ser Leu Gln 100 105 110Phe Val Thr Gln Ser Ser Gly Lys Asn Ile Gly Ser Arg Thr Tyr Leu 115 120 125Met Glu Ser Ser Thr Lys Tyr His Leu Phe Asp Leu Ile Gly Asn Glu 130 135 140Phe Ala Phe Asp Val Asp Leu Ser Lys Leu Pro Cys Gly Leu Asn Gly145 150 155 160Ala Leu Tyr Phe Val Thr Met Asp Ala Asp Gly Gly Met Ala Lys Tyr 165 170 175Ser Thr Asn Thr Ala Gly Ala Glu Tyr Gly Thr Gly Tyr Cys Asp Ser 180 185 190Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Gly Asn Val Glu 195 200 205Gly Trp Thr Pro Ser Thr Asn Asp Ala Asn Ala Gly Val Gly Gly Leu 210 215 220Gly Ser Cys Cys Ser Glu Met Asp Val Trp Glu Ala Asn Ser Met Asp225 230 235 240Met Ala Tyr Thr Pro His Pro Cys Glu Thr Ala Ala Gln His Ser Cys 245 250 255Asn Ala Asp Glu Cys Gly Gly Thr Tyr Ser Ser Ser Arg Tyr Ala Gly 260 265 270Asp Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Phe Arg Met Gly Asn 275 280 285Lys Asp Phe Tyr Gly Ser Gly Asp Thr Val Asp Thr Ser Gln Lys Phe 290 295 300Thr Val Val Thr Gln Phe His Gly Ser Gly Ser Ser Leu Thr Glu Ile305 310 315 320Ser Gln Tyr Tyr Ile Gln Gly Gly Thr Lys Ile Gln Gln Pro Asn Ser 325 330 335Thr Trp Pro Thr Leu Thr Gly Tyr Asn Ser Ile Thr Asp Asp Phe Cys 340 345 350Lys Ala Gln Lys Val Glu Phe Asn Asp Thr Asp Val Phe Ser Glu Lys 355 360 365Gly Gly Leu Ala Gln Met Gly Ala Gly Met Ala Asp Gly Met Val Leu 370 375 380Val Met Ser Leu Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu Asp385 390 395 400Ser Thr Tyr Pro Val Asp Ala Asp Ala Ser Ser Pro Gly Lys Gln Arg 405 410 415Gly Thr Cys Ala Thr Thr Ser Gly Val Pro Ala Asp Val Glu Ser Ser 420 425 430Asp Ala Ser Ala Thr Val Ile Tyr Ser Asn Ile Lys Phe Gly Pro Ile 435 440 445Gly Ala Thr Tyr 45051456PRTCochliobolus carbonum 51Met Tyr Arg Thr Leu Ala Phe Ala Ser Leu Ser Leu Tyr Gly Ala Ala1 5 10 15Arg Ala Gln Gln Val Gly Thr Ser Thr Ala Glu Asn His Pro Lys Leu 20 25 30Thr Trp Gln Thr Cys Thr Gly Thr Gly Gly Thr Asn Cys Ser Asn Lys 35 40 45Ser Gly Ser Val Val Leu Asp Ser Asn Trp Arg Trp Ala His Asn Val 50 55 60Gly Gly Tyr Thr Asn Cys Tyr Thr Gly Asn Ser Trp Ser Thr Gln Tyr65 70 75 80Cys Pro Asp Gly Asp Ser Cys Thr Lys Asn Cys Ala Ile Asp Gly Ala 85 90 95Asp Tyr Ser Gly Thr Tyr Gly Ile Thr Thr Ser Asn Asn Ala Leu Ser 100 105 110Leu Lys Phe Val Thr Lys Gly Ser Phe Ser Ser Asn Ile Gly Ser Arg 115 120 125Thr Tyr Leu Met Glu Thr Asp Thr Lys Tyr Gln Met Phe Asn Leu Ile 130 135 140Asn Lys Glu Phe Thr Phe Asp Val Asp Val Ser Lys Leu Pro Cys Gly145 150 155 160Leu Asn Gly Ala Leu Tyr Phe Val Glu Met Ala Ala Asp Gly Gly Ile 165 170 175Gly Lys Gly Asn Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys 180 185 190Asp Ser Gln Cys Pro His Asp Ile Lys Phe Ile Asn Gly Lys Ala Asn 195 200 205Val Glu Gly Trp Asn Pro Ser Asp Ala Asp Pro Asn Gly Gly Ala Gly 210 215 220Lys Ile Gly Ala Cys Cys Pro Glu Met Asp Ile Trp Glu Ala Asn Ser225 230 235 240Ile Ser Thr Ala Tyr Thr Pro His Pro Cys Arg Gly Val Gly Leu Gln 245 250 255Glu Cys Ser Asp Ala Ala Ser Cys Gly Asp Gly Ser Asn Arg Tyr Asp 260 265 270Gly Gln Cys Asp Lys Asp Gly Cys Asp Phe Asn Ser Tyr Arg Met Gly 275 280 285Val Lys Asp Phe Tyr Gly Pro Gly Ala Thr Leu Asp Thr Thr Lys Lys 290 295 300Met Thr Val Ile Thr Gln Phe Leu Gly Ser Gly Ser Ser Leu Ser Glu305 310 315 320Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Tyr Lys Asn Ser Gln 325 330 335Ser Ala Val Ala Gly Val Thr Gly Asn Ser Ile Thr Glu Ser Phe Cys 340 345 350Thr Ala Gln Lys Lys Ala Phe Gly Asp Thr Ser Ser Phe Ala Ala Leu 355 360 365Gly Gly Leu Asn Glu Met Gly Ala Ser Leu Ala Arg Gly His Val Leu 370 375 380Ile Met Ser Leu Trp Gly Asp His Ala Val Asn Met Leu Trp Leu Asp385 390 395 400Ser Thr Tyr Pro Thr Asp Ala Asp Pro Ser Lys Pro Gly Ala Ala Arg 405 410 415Gly Thr Cys Pro Thr Thr Ser Gly Lys Pro Glu Asp Val Glu Lys Asn 420 425 430Ser Pro Asp Ala Thr Val Val Phe Ser Asn Ile Lys Phe Gly Pro Ile 435 440 445Gly Ser Thr Phe Ala Gln Pro Ala 450 45552525PRTHumicola grisea 52Met Arg Thr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Ser Ala1 5 10 15Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro Ser Leu 20 25 30Ser Trp Asn Lys Cys Thr Ala Gly Gly Gln Cys Gln Thr Val Gln Ala 35 40 45Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp Thr His Gln Val Ser Gly 50 55 60 Ser Thr Asn Cys Tyr Thr Gly Asn Lys Trp Asp Thr Ser Ile Cys Thr65 70 75 80Asp Ala Lys Ser Cys Ala Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr 85 90 95Thr Ser Thr Tyr Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 100 105 110Phe Val Thr Lys Gly Gln His Ser Thr Asn Val Gly Ser Arg Thr Tyr 115 120 125Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu Leu Leu Gly Asn 130 135 140Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly Leu Asn145 150 155 160Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu Ser Arg 165 170 175Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185 190Ala Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile 195 200 205Glu Gly Trp Thr Gly Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 210 215 220Tyr Gly Thr Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met225 230 235 240Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 245 250 255Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg Tyr Ala 260 265 270Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly 275 280 285Asn Lys Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys 290 295 300Ile Thr Val Val Thr Gln Phe Leu Lys Asp Ala Asn Gly Asp Leu Gly305 310 315 320Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Pro Asn Ser 325 330 335Glu Ser Thr Ile Pro Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp 340 345 350Cys Asp Arg Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 355 360 365Lys Gly Gly Met Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val 370 375 380Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met Leu Trp Leu385 390 395 400Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly Ala Glu Arg 405 410 415Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu 420 425 430Ala Pro Asn Ser Asn Val Val Phe Ser Asn Ile Arg Phe Gly Pro Ile 435 440 445Gly Ser Thr Val Ala Gly Leu Pro Gly Ala Gly Asn Gly Gly Asn Asn 450 455 460Gly Gly Asn Pro Pro Pro Pro Thr Thr Thr Thr Ser Ser Ala Pro Ala465 470 475 480Thr Thr Thr Thr Ala Ser Ala Gly Pro Lys Ala Gly Arg Trp Gln Gln 485 490 495Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 500 505 510Ile Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 515 520 52553525PRTHumicola grisea 53Met Arg Thr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Ser Ala1 5 10 15Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro Ser Leu 20 25 30 Ser Trp Lys Lys Cys Thr Ala Gly Gly Gln Cys Gln Thr Val Gln Ala 35 40 45Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp Thr His Gln Val Ser Gly 50 55 60Ser Thr Asn Cys Tyr Thr Gly Asn Lys Trp Asp Thr Ser Ile Cys Thr65 70 75 80Asp Ala Lys Ser Cys Ala Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr 85 90 95Thr Ser Thr Tyr Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 100 105 110Phe Val Thr Lys Gly Gln Tyr Ser Thr Asn Val Gly Ser Arg Thr Tyr 115 120 125Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu Leu Leu Gly Asn 130 135 140Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile

Gly Cys Gly Leu Asn145 150 155 160Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu Ser Arg 165 170 175Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185 190Ala Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile 195 200 205Glu Gly Trp Thr Gly Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 210 215 220Tyr Gly Thr Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met225 230 235 240Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 245 250 255Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg Tyr Ala 260 265 270Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly 275 280 285Asn Lys Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys 290 295 300Ile Thr Val Val Thr Gln Phe Leu Lys Asp Ala Asn Gly Asp Leu Gly305 310 315 320Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Pro Asn Ser 325 330 335Glu Ser Thr Ile Pro Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp 340 345 350Cys Asp Arg Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 355 360 365Lys Gly Gly Met Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val 370 375 380Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met Leu Trp Leu385 390 395 400Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly Ala Glu Arg 405 410 415Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu 420 425 430Ala Pro Asn Ser Asn Val Val Phe Ser Asn Ile Arg Phe Gly Pro Ile 435 440 445Gly Ser Thr Val Ala Gly Leu Pro Gly Ala Gly Asn Gly Gly Asn Asn 450 455 460Gly Gly Asn Pro Pro Pro Pro Thr Thr Thr Thr Ser Ser Ala Pro Ala465 470 475 480Thr Thr Thr Thr Ala Ser Ala Gly Pro Lys Ala Gly Arg Trp Gln Gln 485 490 495Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 500 505 510Thr Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 515 520 52554514PRTFusarium oxysporum 54Met Tyr Arg Ile Val Ala Thr Ala Ser Ala Leu Ile Ala Ala Ala Arg1 5 10 15Ala Gln Gln Val Cys Ser Leu Asn Thr Glu Thr Lys Pro Ala Leu Thr 20 25 30Trp Ser Lys Cys Thr Ser Ser Gly Cys Ser Asp Val Lys Gly Ser Val 35 40 45Val Ile Asp Ala Asn Trp Arg Trp Thr His Gln Thr Ser Gly Ser Thr 50 55 60Asn Cys Tyr Thr Gly Asn Lys Trp Asp Thr Ser Ile Cys Thr Asp Gly65 70 75 80Lys Thr Cys Ala Glu Lys Cys Cys Leu Asp Gly Ala Asp Tyr Ser Gly 85 90 95Thr Tyr Gly Ile Thr Ser Ser Gly Asn Gln Leu Ser Leu Gly Phe Val 100 105 110Thr Asn Gly Pro Tyr Ser Lys Asn Ile Gly Ser Arg Thr Tyr Leu Met 115 120 125Glu Asn Glu Asn Thr Tyr Gln Met Phe Gln Leu Leu Gly Asn Glu Phe 130 135 140Thr Phe Asp Val Asp Val Ser Gly Ile Gly Cys Gly Leu Asn Gly Ala145 150 155 160Pro His Phe Val Ser Met Asp Glu Asp Gly Gly Lys Ala Lys Tyr Ser 165 170 175Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ala Gln 180 185 190Cys Pro Arg Asp Val Lys Phe Ile Asn Gly Val Ala Asn Ser Glu Gly 195 200 205Trp Lys Pro Ser Asp Ser Asp Val Asn Ala Gly Val Gly Asn Leu Gly 210 215 220Thr Cys Cys Pro Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Thr225 230 235 240Ala Phe Thr Pro His Pro Cys Thr Lys Leu Thr Gln His Ser Cys Thr 245 250 255Gly Asp Ser Cys Gly Gly Thr Tyr Ser Ser Asp Arg Tyr Gly Gly Thr 260 265 270Cys Asp Ala Asp Gly Cys Asp Phe Asn Ala Tyr Arg Gln Gly Asn Lys 275 280 285Thr Phe Tyr Gly Pro Gly Ser Asn Phe Asn Ile Asp Thr Thr Lys Lys 290 295 300Met Thr Val Val Thr Gln Phe His Lys Gly Ser Asn Gly Arg Leu Ser305 310 315 320Glu Ile Thr Arg Leu Tyr Val Gln Asn Gly Lys Val Ile Ala Asn Ser 325 330 335Glu Ser Lys Ile Ala Gly Asn Pro Gly Ser Ser Leu Thr Ser Asp Phe 340 345 350Cys Ser Lys Gln Lys Ser Val Phe Gly Asp Ile Asp Asp Phe Ser Lys 355 360 365Lys Gly Gly Trp Asn Gly Met Ser Asp Ala Leu Ser Ala Pro Met Val 370 375 380Leu Val Met Ser Leu Trp His Asp His His Ser Asn Met Leu Trp Leu385 390 395 400Asp Ser Thr Tyr Pro Thr Asp Ser Thr Lys Val Gly Ser Gln Arg Gly 405 410 415Ser Cys Ala Thr Thr Ser Gly Lys Pro Ser Asp Leu Glu Arg Asp Val 420 425 430Pro Asn Ser Lys Val Ser Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly 435 440 445Ser Thr Tyr Lys Ser Asp Gly Thr Thr Pro Asn Pro Pro Ala Ser Ser 450 455 460Ser Thr Thr Gly Ser Ser Thr Pro Thr Asn Pro Pro Ala Gly Ser Val465 470 475 480Asp Gln Trp Gly Gln Cys Gly Gly Gln Asn Tyr Ser Gly Pro Thr Thr 485 490 495Cys Lys Ser Pro Phe Thr Cys Lys Lys Ile Asn Asp Phe Tyr Ser Gln 500 505 510Cys Gln55456PRTClaviceps purpurea 55Met His Pro Ser Leu Gln Thr Ile Leu Leu Ser Ala Leu Phe Thr Thr1 5 10 15Ala His Ala Gln Gln Ala Cys Ser Ser Lys Pro Glu Thr His Pro Pro 20 25 30Leu Ser Trp Ser Arg Cys Ser Arg Ser Gly Cys Arg Ser Val Gln Gly 35 40 45Ala Val Thr Val Asp Ala Asn Trp Leu Trp Thr Thr Val Asp Gly Ser 50 55 60Gln Asn Cys Tyr Thr Gly Asn Arg Trp Asp Thr Ser Ile Cys Ser Ser65 70 75 80Glu Lys Thr Cys Ser Glu Ser Cys Cys Ile Asp Gly Ala Asp Tyr Ala 85 90 95Gly Thr Tyr Gly Val Thr Thr Thr Gly Asp Ala Leu Ser Leu Lys Phe 100 105 110Val Gln Gln Gly Pro Tyr Ser Lys Asn Val Gly Ser Arg Leu Tyr Leu 115 120 125Met Lys Asp Glu Ser Arg Tyr Glu Met Phe Thr Leu Leu Gly Asn Glu 130 135 140Phe Thr Phe Asp Val Asp Val Ser Lys Leu Gly Cys Gly Leu Asn Gly145 150 155 160Ala Leu Tyr Phe Val Ser Met Asp Glu Asp Gly Gly Met Lys Arg Phe 165 170 175Pro Met Asn Lys Ala Gly Ala Lys Phe Gly Thr Gly Tyr Cys Asp Ser 180 185 190Gln Cys Pro Arg Asp Val Lys Phe Ile Asn Gly Met Ala Asn Ser Lys 195 200 205Asp Trp Ile Pro Ser Lys Ser Asp Ala Asn Ala Gly Ile Gly Ser Leu 210 215 220Gly Ala Cys Cys Arg Glu Met Asp Ile Trp Glu Ala Asn Asn Ile Ala225 230 235 240Ser Ala Phe Thr Pro His Pro Cys Lys Asn Ser Ala Tyr His Ser Cys 245 250 255Thr Gly Asp Gly Cys Gly Gly Thr Tyr Ser Lys Asn Arg Tyr Ser Gly 260 265 270Asp Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Leu Gly Asn 275 280 285Thr Thr Phe Tyr Gly Pro Gly Pro Lys Phe Thr Ile Asp Thr Thr Arg 290 295 300Lys Ile Ser Val Val Thr Gln Phe Leu Lys Gly Arg Asp Gly Ser Leu305 310 315 320Arg Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Asn 325 330 335Ser Val Ser Arg Val Arg Gly Val Pro Gly Asn Ser Ile Thr Gln Gly 340 345 350Phe Cys Asn Ala Gln Lys Lys Met Phe Gly Ala His Glu Ser Phe Asn 355 360 365Ala Lys Gly Gly Met Lys Gly Met Ser Ala Ala Val Ser Lys Pro Met 370 375 380Val Leu Val Met Ser Leu Trp Asp Asp His Asn Ser Asn Met Leu Trp385 390 395 400Leu Asp Ser Thr Tyr Pro Thr Asn Ser Arg Gln Arg Gly Ser Lys Arg 405 410 415Gly Ser Cys Pro Ala Ser Ser Gly Arg Pro Thr Asp Val Glu Ser Ser 420 425 430Ala Pro Asp Ser Thr Val Val Phe Ser Asn Ile Lys Phe Gly Pro Ile 435 440 445Gly Ser Thr Phe Ser Arg Gly Lys 450 45556451PRTHumicola grisea var. thermoidea 56Met Gln Ile Lys Ser Tyr Ile Gln Tyr Leu Ala Ala Ala Leu Pro Leu1 5 10 15Leu Ser Ser Val Ala Ala Gln Gln Ala Gly Thr Ile Thr Ala Glu Asn 20 25 30His Pro Arg Met Thr Trp Lys Arg Cys Ser Gly Pro Gly Asn Cys Gln 35 40 45Thr Val Gln Gly Glu Val Val Ile Asp Ala Asn Trp Arg Trp Leu His 50 55 60Asn Asn Gly Gln Asn Cys Tyr Glu Gly Asn Lys Trp Thr Ser Gln Cys65 70 75 80Ser Ser Ala Thr Asp Cys Ala Gln Arg Cys Ala Leu Asp Gly Ala Asn 85 90 95Tyr Gln Ser Thr Tyr Gly Ala Ser Thr Ser Gly Asp Ser Leu Thr Leu 100 105 110Lys Phe Val Thr Lys His Glu Tyr Gly Thr Asn Ile Gly Ser Arg Phe 115 120 125Tyr Leu Met Ala Asn Gln Asn Lys Tyr Gln Met Phe Thr Leu Met Asn 130 135 140Asn Glu Phe Ala Phe Asp Val Asp Leu Ser Lys Val Glu Cys Gly Ile145 150 155 160Asn Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly Met Ala 165 170 175Ser Tyr Pro Ser Asn Arg Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys 180 185 190Asp Ala Gln Cys Ala Arg Asp Leu Lys Phe Ile Gly Gly Lys Ala Asn 195 200 205Ile Glu Gly Trp Arg Pro Ser Thr Asn Asp Pro Asn Ala Gly Val Gly 210 215 220Pro Met Gly Ala Cys Cys Ala Glu Ile Asp Val Trp Glu Ser Asn Ala225 230 235 240Tyr Ala Tyr Ala Phe Thr Pro His Ala Cys Gly Ser Lys Asn Arg Tyr 245 250 255His Ile Cys Glu Thr Asn Asn Cys Gly Gly Thr Tyr Ser Asp Asp Arg 260 265 270Phe Ala Gly Tyr Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr Arg 275 280 285Met Gly Asn Lys Asp Phe Tyr Gly Lys Gly Lys Thr Val Asp Thr Asn 290 295 300Arg Lys Phe Thr Val Val Ser Arg Phe Glu Arg Asn Arg Leu Ser Gln305 310 315 320Phe Phe Val Gln Asp Gly Arg Lys Ile Glu Val Pro Pro Pro Thr Trp 325 330 335Pro Gly Leu Pro Asn Ser Ala Asp Ile Thr Pro Glu Leu Cys Asp Ala 340 345 350Gln Phe Arg Val Phe Asp Asp Arg Asn Arg Phe Ala Glu Thr Gly Gly 355 360 365Phe Asp Ala Leu Asn Glu Ala Leu Thr Ile Pro Met Val Leu Val Met 370 375 380Ser Ile Trp Asp Asp His His Ser Asn Met Leu Trp Leu Asp Ser Ser385 390 395 400Tyr Pro Pro Glu Lys Ala Gly Leu Pro Gly Gly Asp Arg Gly Pro Cys 405 410 415Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Gln Tyr Pro Asn 420 425 430Ala Gln Val Val Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr 435 440 445Val Asn Val 45057451PRTHumicola grisea var. thermoidea 57Met Gln Ile Lys Ser Tyr Ile Gln Tyr Leu Ala Ala Ala Leu Pro Leu1 5 10 15Leu Ser Ser Val Ala Ala Gln Gln Ala Gly Thr Ile Thr Ala Glu Asn 20 25 30His Pro Arg Met Thr Trp Lys Arg Cys Ser Gly Pro Gly Asn Cys Gln 35 40 45Thr Val Gln Gly Glu Val Val Ile Asp Ala Asn Trp Arg Trp Leu His 50 55 60Asn Asn Gly Gln Asn Cys Tyr Glu Gly Asn Lys Trp Thr Ser Gln Cys65 70 75 80Ser Ser Ala Thr Asp Cys Ala Gln Arg Cys Ala Leu Asp Gly Ala Asn 85 90 95Tyr Gln Ser Thr Tyr Gly Ala Ser Thr Ser Gly Asp Ser Leu Thr Leu 100 105 110Lys Phe Val Thr Lys His Glu Tyr Gly Thr Asn Ile Gly Ser Arg Phe 115 120 125Tyr Leu Met Ala Asn Gln Asn Lys Tyr Gln Met Phe Thr Leu Met Asn 130 135 140Asn Glu Phe Ala Phe Asp Val Asp Leu Ser Lys Val Glu Cys Gly Ile145 150 155 160Asn Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly Met Ala 165 170 175Ser Tyr Pro Ser Asn Arg Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys 180 185 190Asp Ala Gln Cys Ala Arg Asp Leu Lys Phe Ile Gly Gly Lys Ala Asn 195 200 205Ile Glu Gly Trp Arg Pro Ser Thr Asn Asp Pro Asn Ala Gly Val Gly 210 215 220Pro Met Gly Ala Cys Cys Ala Glu Ile Asp Val Trp Glu Ser Asn Ala225 230 235 240Tyr Ala Tyr Ala Phe Thr Pro His Ala Cys Gly Ser Lys Asn Arg Tyr 245 250 255His Ile Cys Glu Thr Asn Asn Cys Gly Gly Thr Tyr Ser Asp Asp Arg 260 265 270Phe Ala Gly Tyr Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr Arg 275 280 285Met Gly Asn Lys Asp Phe Tyr Gly Lys Gly Lys Thr Val Asp Thr Asn 290 295 300Arg Lys Phe Thr Val Val Ser Arg Phe Glu Arg Asn Arg Leu Ser Gln305 310 315 320Phe Phe Val Gln Asp Gly Arg Lys Ile Glu Val Pro Pro Pro Thr Trp 325 330 335Pro Gly Leu Pro Asn Ser Ala Asp Ile Thr Pro Glu Leu Cys Asp Ala 340 345 350Gln Phe Arg Val Phe Asp Asp Arg Asn Arg Phe Ala Glu Thr Gly Gly 355 360 365Phe Asp Ala Leu Asn Glu Ala Leu Thr Ile Pro Met Val Leu Val Met 370 375 380Ser Ile Trp Asp Asp His His Ser Asn Met Leu Trp Leu Asp Ser Ser385 390 395 400Tyr Pro Pro Glu Lys Ala Gly Leu Pro Gly Gly Asp Arg Gly Pro Cys 405 410 415Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Gln Tyr Pro Asp 420 425 430Ala Gln Val Val Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr 435 440 445Val Asn Val 45058447PRTLeptosphaeria maculans 58Met Leu Ser Ala Ser Lys Ala Ala Ala Ile Leu Ala Phe Cys Ala His1 5 10 15Thr Ala Ser Ala Trp Val Val Gly Asp Gln Gln Thr Glu Thr His Pro 20 25 30Lys Leu Asn Trp Gln Arg Cys Thr Gly Lys Gly Arg Ser Ser Cys Thr 35 40 45Asn Val Asn Gly Glu Val Val Ile Asp Ala Asn Trp Arg Trp Leu Ala 50 55 60His Arg Ser Gly Tyr Thr Asn Cys Tyr Thr Gly Ser Glu Trp Asn Gln65 70 75 80Ser Ala Cys Pro Asn Asn Glu Ala Cys Thr Lys Asn Cys Ala Ile Glu 85 90 95Gly Ser Asp Tyr Ala Gly Thr Tyr Gly Ile Thr Thr Ser Gly Asn Gln 100 105 110Met Asn Ile Lys Phe Ile Thr Lys Arg Pro Tyr Ser Thr Asn Ile Gly 115 120 125Ala Arg Thr Tyr Leu Met Lys Asp Glu Gln Asn Tyr Glu Met Phe Gln 130 135 140Leu Ile Gly Asn Glu Phe Thr Phe Asp Val Asp Leu Ser Gln Arg Cys145 150 155 160Gly Met Asn Gly Ala Leu Tyr Phe Val Ser Met Pro Gln Lys Gly Gln 165 170 175Gly Ala Pro Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ala Gln Cys 180 185 190Ala Arg Asp Leu Lys Phe Val Arg Gly Ser Ala Asn Ala Glu Gly Trp 195

200 205Thr Lys Ser Ala Ser Asp Pro Asn Ser Gly Val Gly Lys Lys Gly Ala 210 215 220Cys Cys Ala Gln Met Asp Val Trp Glu Ala Asn Ser Ala Ala Thr Ala225 230 235 240Leu Thr Pro His Ser Cys Gln Pro Ala Gly Tyr Ser Val Cys Glu Asp 245 250 255Thr Asn Cys Gly Gly Thr Tyr Ser Glu Asp Arg Tyr Ala Gly Thr Cys 260 265 270Asp Ala Asn Gly Cys Asp Phe Asn Pro Phe Arg Val Gly Val Lys Asp 275 280 285Phe Tyr Gly Lys Gly Lys Thr Val Asp Thr Thr Lys Lys Met Thr Val 290 295 300Val Thr Gln Phe Val Gly Ser Gly Asn Gln Leu Ser Glu Ile Lys Arg305 310 315 320Phe Tyr Val Gln Asp Gly Lys Val Ile Ala Asn Pro Glu Pro Thr Ile 325 330 335Pro Gly Met Glu Trp Cys Asn Thr Gln Lys Lys Val Phe Gln Glu Glu 340 345 350Ala Tyr Pro Phe Asn Glu Phe Gly Gly Met Ala Ser Met Ser Glu Gly 355 360 365Met Ser Gln Gly Met Val Leu Val Met Ser Leu Trp Asp Asp His Tyr 370 375 380Ala Asn Met Leu Trp Leu Asp Ser Asn Trp Pro Arg Glu Ala Asp Pro385 390 395 400Ala Lys Pro Gly Val Ala Arg Arg Asp Cys Pro Thr Ser Gly Gly Lys 405 410 415Pro Ser Glu Val Glu Ala Ala Asn Pro Asn Ala Gln Val Met Phe Ser 420 425 430 Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Phe Ala His Ala Ala 435 440 44559516PRTNeurospora crassa 59Met Arg Ala Ser Leu Leu Ala Phe Ser Leu Ala Ala Ala Val Ala Gly1 5 10 15Gly Gln Gln Ala Gly Thr Leu Thr Ala Lys Arg His Pro Ser Leu Thr 20 25 30Trp Gln Lys Cys Thr Arg Gly Gly Cys Pro Thr Leu Asn Thr Thr Met 35 40 45Val Leu Asp Ala Asn Trp Arg Trp Thr His Ala Thr Ser Gly Ser Thr 50 55 60Lys Cys Tyr Thr Gly Asn Lys Trp Gln Ala Thr Leu Cys Pro Asp Gly65 70 75 80Lys Ser Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr Thr Gly 85 90 95Thr Tyr Gly Ile Thr Gly Ser Gly Trp Ser Leu Thr Leu Gln Phe Val 100 105 110Thr Asp Asn Val Gly Ala Arg Ala Tyr Leu Met Ala Asp Asp Thr Gln 115 120 125Tyr Gln Met Leu Glu Leu Leu Asn Gln Glu Leu Trp Phe Asp Val Asp 130 135 140Met Ser Asn Ile Pro Cys Gly Leu Asn Gly Ala Leu Tyr Leu Ser Ala145 150 155 160Met Asp Ala Asp Gly Gly Met Arg Lys Tyr Pro Thr Asn Lys Ala Gly 165 170 175Ala Lys Tyr Ala Thr Gly Tyr Cys Asp Ala Gln Cys Pro Arg Asp Leu 180 185 190Lys Tyr Ile Asn Gly Ile Ala Asn Val Glu Gly Trp Thr Pro Ser Thr 195 200 205Asn Asp Ala Asn Gly Ile Gly Asp His Gly Ser Cys Cys Ser Glu Met 210 215 220Asp Ile Trp Glu Ala Asn Lys Val Ser Thr Ala Phe Thr Pro His Pro225 230 235 240Cys Thr Thr Ile Glu Gln His Met Cys Glu Gly Asp Ser Cys Gly Gly 245 250 255Thr Tyr Ser Asp Asp Arg Tyr Gly Val Leu Cys Asp Ala Asp Gly Cys 260 265 270Asp Phe Asn Ser Tyr Arg Met Gly Asn Thr Thr Phe Tyr Gly Glu Gly 275 280 285Lys Thr Val Asp Thr Ser Ser Lys Phe Thr Val Val Thr Gln Phe Ile 290 295 300Lys Asp Ser Ala Gly Asp Leu Ala Glu Ile Lys Ala Phe Tyr Val Gln305 310 315 320Asn Gly Lys Val Ile Glu Asn Ser Gln Ser Asn Val Asp Gly Val Ser 325 330 335Gly Asn Ser Ile Thr Gln Ser Phe Cys Lys Ser Gln Lys Thr Ala Phe 340 345 350Gly Asp Ile Asp Asp Phe Asn Lys Lys Gly Gly Leu Lys Gln Met Gly 355 360 365Lys Ala Leu Ala Gln Ala Met Val Leu Val Met Ser Ile Trp Asp Asp 370 375 380His Ala Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Val Pro Lys385 390 395 400Val Pro Gly Ala Tyr Arg Gly Ser Gly Pro Thr Thr Ser Gly Val Pro 405 410 415Ala Glu Val Asp Ala Asn Ala Pro Asn Ser Lys Val Ala Phe Ser Asn 420 425 430Ile Lys Phe Gly His Leu Gly Ile Ser Pro Phe Ser Gly Gly Ser Ser 435 440 445Gly Thr Pro Pro Ser Asn Pro Ser Ser Ser Ala Ser Pro Thr Ser Ser 450 455 460Thr Ala Lys Pro Ser Ser Thr Ser Thr Ala Ser Asn Pro Ser Gly Thr465 470 475 480Gly Ala Ala His Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser Gly Pro 485 490 495Thr Thr Cys Pro Glu Pro Tyr Thr Cys Ala Lys Asp His Asp Ile Tyr 500 505 510Ser Gln Cys Val 51560540PRTAspergillus aculeatus 60Met Val Asp Ser Phe Ser Ile Tyr Lys Thr Ala Leu Leu Leu Ser Met1 5 10 15Leu Ala Thr Ser Asn Ala Gln Gln Val Gly Thr Tyr Thr Ala Glu Thr 20 25 30His Pro Ser Leu Thr Trp Gln Thr Cys Ser Gly Ser Gly Ser Cys Thr 35 40 45Thr Thr Ser Gly Ser Val Val Ile Asp Ala Asn Trp Arg Trp Val His 50 55 60Glu Val Gly Gly Tyr Thr Asn Cys Tyr Ser Gly Asn Thr Trp Asp Ser65 70 75 80Ser Ile Cys Ser Thr Asp Thr Thr Cys Ala Ser Glu Cys Ala Leu Glu 85 90 95Gly Ala Thr Tyr Glu Ser Thr Tyr Gly Val Thr Thr Ser Gly Ser Ser 100 105 110Leu Arg Leu Asn Phe Val Thr Thr Ala Ser Gln Lys Asn Ile Gly Ser 115 120 125Arg Leu Tyr Leu Leu Ala Asp Asp Ser Thr Tyr Glu Thr Phe Lys Leu 130 135 140Phe Asn Arg Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys145 150 155 160Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly 165 170 175Val Ser Arg Phe Pro Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly 180 185 190Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asp Gly Gln 195 200 205Ala Asn Ile Glu Gly Trp Glu Pro Ser Ser Thr Asp Val Asn Ala Gly 210 215 220Thr Gly Asn His Gly Ser Cys Cys Pro Glu Met Asp Ile Trp Glu Ala225 230 235 240Asn Ser Ile Ser Ser Ala Phe Thr Ala His Pro Cys Asp Ser Val Gln 245 250 255Gln Thr Met Cys Thr Gly Asp Thr Cys Gly Gly Thr Tyr Ser Asp Thr 260 265 270Thr Asp Arg Tyr Ser Gly Thr Cys Asp Pro Asp Gly Cys Asp Phe Asn 275 280 285Pro Tyr Arg Phe Gly Asn Thr Asn Phe Tyr Gly Pro Gly Lys Thr Val 290 295 300Asp Asn Ser Lys Pro Phe Thr Val Val Thr Gln Phe Ile Thr His Asp305 310 315 320Gly Thr Asp Thr Gly Thr Leu Thr Glu Ile Arg Arg Leu Tyr Val Gln 325 330 335Asn Gly Val Val Ile Gly Asn Gly Pro Ser Thr Tyr Thr Ala Ala Ser 340 345 350Gly Asn Ser Ile Thr Glu Ser Phe Cys Lys Ala Glu Lys Thr Leu Phe 355 360 365Gly Asp Thr Asn Val Phe Glu Thr His Gly Gly Leu Ser Ala Met Gly 370 375 380Asp Ala Leu Gly Asp Gly Met Val Leu Val Leu Ser Leu Trp Asp Asp385 390 395 400His Ala Ala Asp Met Leu Trp Leu Asp Ser Asp Tyr Pro Thr Thr Ser 405 410 415Cys Ala Ser Ser Pro Gly Val Ala Arg Gly Thr Cys Pro Thr Thr Thr 420 425 430Gly Asn Ala Thr Tyr Val Glu Ala Asn Tyr Pro Asn Ser Tyr Val Thr 435 440 445Tyr Ser Asn Ile Lys Phe Gly Thr Leu Asn Ser Thr Tyr Ser Gly Thr 450 455 460Ser Ser Gly Gly Ser Ser Ser Ser Ser Thr Thr Leu Thr Thr Lys Ala465 470 475 480Ser Thr Ser Thr Thr Ser Ser Lys Thr Thr Thr Thr Thr Ser Lys Thr 485 490 495Ser Thr Thr Ser Ser Ser Ser Thr Asn Val Ala Gln Leu Tyr Gly Gln 500 505 510Cys Gly Gly Gln Gly Trp Thr Gly Pro Thr Thr Cys Ala Ser Gly Thr 515 520 525Cys Thr Lys Gln Asn Asp Tyr Tyr Ser Gln Cys Leu 530 535 54061536PRTAspergillus niger 61Met Ser Ser Phe Gln Ile Tyr Arg Ala Ala Leu Leu Leu Ser Ile Leu1 5 10 15Ala Thr Ala Asn Ala Gln Gln Val Gly Thr Tyr Thr Thr Glu Thr His 20 25 30Pro Ser Leu Thr Trp Gln Thr Cys Thr Ser Asp Gly Ser Cys Thr Thr 35 40 45 Asn Asp Gly Glu Val Val Ile Asp Ala Asn Trp Arg Trp Val His Ser 50 55 60Thr Ser Ser Ala Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser65 70 75 80Ile Cys Thr Asp Asp Val Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly 85 90 95Ala Thr Tyr Glu Ala Thr Tyr Gly Val Thr Thr Ser Gly Ser Glu Leu 100 105 110Arg Leu Asn Phe Val Thr Gln Gly Ser Ser Lys Asn Ile Gly Ser Arg 115 120 125Leu Tyr Leu Met Ser Asp Asp Ser Asn Tyr Glu Leu Phe Lys Leu Leu 130 135 140Gly Gln Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys Gly145 150 155 160Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp Ala Asp Gly Gly Thr 165 170 175Ser Glu Tyr Ser Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr 180 185 190Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Ala 195 200 205Asn Cys Asp Gly Trp Glu Pro Ser Ser Asn Asn Val Asn Thr Gly Val 210 215 220Gly Asp His Gly Ser Cys Cys Ala Glu Met Asp Val Trp Glu Ala Asn225 230 235 240Ser Ile Ser Asn Ala Phe Thr Ala His Pro Cys Asp Ser Val Ser Gln 245 250 255Thr Met Cys Asp Gly Asp Ser Cys Gly Gly Thr Tyr Ser Ala Ser Gly 260 265 270Asp Arg Tyr Ser Gly Thr Cys Asp Pro Asp Gly Cys Asp Tyr Asn Pro 275 280 285Tyr Arg Leu Gly Asn Thr Asp Phe Tyr Gly Pro Gly Leu Thr Val Asp 290 295 300Thr Asn Ser Pro Phe Thr Val Val Thr Gln Phe Ile Thr Asp Asp Gly305 310 315 320Thr Ser Ser Gly Thr Leu Thr Glu Ile Lys Arg Leu Tyr Val Gln Asn 325 330 335Gly Glu Val Ile Ala Asn Gly Ala Ser Thr Tyr Ser Ser Val Asn Gly 340 345 350Ser Ser Ile Thr Ser Ala Phe Cys Glu Ser Glu Lys Thr Leu Phe Gly 355 360 365Asp Glu Asn Val Phe Asp Lys His Gly Gly Leu Glu Gly Met Gly Glu 370 375 380Ala Met Ala Lys Gly Met Val Leu Val Leu Ser Leu Trp Asp Asp Tyr385 390 395 400Ala Ala Asp Met Leu Trp Leu Asp Ser Asp Tyr Pro Val Asn Ser Ser 405 410 415Ala Ser Thr Pro Gly Val Ala Arg Gly Thr Cys Ser Thr Asp Ser Gly 420 425 430Val Pro Ala Thr Val Glu Ala Glu Ser Pro Asn Ala Tyr Val Thr Tyr 435 440 445Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Tyr Ser Ser Gly Ser 450 455 460Ser Ser Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Thr Thr Thr Lys465 470 475 480Ala Thr Ser Thr Thr Leu Lys Thr Thr Ser Thr Thr Ser Ser Gly Ser 485 490 495Ser Ser Thr Ser Ala Ala Gln Ala Tyr Gly Gln Cys Gly Gly Gln Gly 500 505 510Trp Thr Gly Pro Thr Thr Cys Val Ser Gly Tyr Thr Cys Thr Tyr Glu 515 520 525Asn Ala Tyr Tyr Ser Gln Cys Leu 530 53562537PRTPenicillum janthinellumVARIANT48, 64Xaa = Any Amino Acid 62Met Lys Gly Ser Ile Ser Tyr Gln Ile Tyr Lys Gly Ala Leu Leu Leu1 5 10 15Ser Ala Leu Leu Asn Ser Val Ser Ala Gln Gln Val Gly Thr Leu Thr 20 25 30Ala Glu Thr His Pro Ala Leu Thr Trp Ser Lys Cys Thr Ala Gly Xaa 35 40 45Cys Ser Gln Val Ser Gly Ser Val Val Ile Asp Ala Asn Trp Pro Xaa 50 55 60Val His Ser Thr Ser Gly Ser Thr Asn Cys Tyr Thr Gly Asn Thr Trp65 70 75 80Asp Ala Thr Leu Cys Pro Asp Asp Val Thr Cys Ala Ala Asn Cys Ala 85 90 95Val Asp Gly Ala Arg Arg Gln His Leu Arg Val Thr Thr Ser Gly Asn 100 105 110Ser Leu Arg Ile Asn Phe Val Thr Thr Ala Ser Gln Lys Asn Ile Gly 115 120 125Ser Arg Leu Tyr Leu Leu Glu Asn Asp Thr Thr Tyr Gln Lys Phe Asn 130 135 140Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Pro145 150 155 160Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Asp Met Asp Ala Asp Gly 165 170 175Gly Met Ala Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr 180 185 190Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly 195 200 205Gln Ala Asn Val Asp Gly Trp Thr Pro Ser Lys Asn Asp Val Asn Ser 210 215 220Gly Ile Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp Ile Trp Glu225 230 235 240Ala Asn Ser Ile Ser Asn Ala Val Thr Pro His Pro Cys Asp Thr Pro 245 250 255Ser Gln Thr Met Cys Thr Gly Gln Arg Cys Gly Gly Thr Tyr Ser Thr 260 265 270Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Phe Asn Pro 275 280 285Tyr Arg Met Gly Val Thr Asn Phe Tyr Gly Pro Gly Glu Thr Ile Asp 290 295 300Thr Lys Ser Pro Phe Thr Val Val Thr Gln Phe Leu Thr Asn Asp Gly305 310 315 320Thr Ser Thr Gly Thr Leu Ser Glu Ile Lys Arg Phe Tyr Val Gln Gly 325 330 335Gly Lys Val Ile Gly Asn Pro Gln Ser Thr Ile Val Gly Val Ser Gly 340 345 350Asn Ser Ile Thr Asp Ser Trp Cys Asn Ala Gln Lys Ser Ala Phe Gly 355 360 365Asp Thr Asn Glu Phe Ser Lys His Gly Gly Met Ala Gly Met Gly Ala 370 375 380Gly Leu Ala Asp Gly Met Val Leu Val Met Ser Leu Trp Asp Asp His385 390 395 400Ala Ser Asp Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn Ala Thr 405 410 415Ser Thr Thr Pro Gly Ala Lys Arg Gly Thr Cys Asp Ile Ser Arg Arg 420 425 430Pro Asn Thr Val Glu Ser Thr Tyr Pro Asn Ala Tyr Val Ile Tyr Ser 435 440 445Asn Ile Lys Thr Gly Pro Leu Asn Ser Thr Phe Thr Gly Gly Thr Thr 450 455 460Ser Ser Ser Ser Thr Thr Thr Thr Thr Ser Lys Ser Thr Ser Thr Ser465 470 475 480Ser Ser Ser Lys Thr Thr Thr Thr Val Thr Thr Thr Thr Thr Ser Ser 485 490 495Gly Ser Ser Gly Thr Gly Ala Arg Asp Trp Ala Gln Cys Gly Gly Asn 500 505 510Gly Trp Thr Gly Pro Thr Thr Cys Val Ser Pro Tyr Thr Cys Thr Lys 515 520 525Gln Asn Asp Trp Tyr Ser Gln Cys Leu 530 53563452PRTAspergillus niger 63Met His Gln Arg Ala Leu Leu Phe Ser Ala Leu Leu Thr Ala Val Arg1 5 10 15Ala Gln Gln Ala Gly Thr Leu Thr Glu Glu Val His Pro Ser Leu Thr 20 25 30Trp Gln Lys Cys Thr Ser Glu Gly Ser Cys Thr Glu Gln Ser Gly Ser 35 40 45Val Val Ile Asp Ser Asn Trp Arg Trp Thr His Ser Val Asn Asp Ser 50 55 60Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Ala Thr Leu Cys Pro Asp65 70

75 80Asp Glu Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu 85 90 95Ser Thr Tyr Gly Val Thr Thr Asp Gly Asp Ser Leu Thr Leu Lys Phe 100 105 110Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Met Asp Thr Ser 115 120 125Asp Glu Gly Tyr Gln Thr Phe Asn Leu Leu Asp Ala Glu Phe Thr Phe 130 135 140Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr145 150 155 160Phe Thr Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Ala Asn 165 170 175Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Arg Asp Leu Lys Phe Ile Asp Gly Gln Ala Asn Val Asp Gly Trp Glu 195 200 205Pro Ser Ser Asn Asn Asp Asn Thr Gly Ile Gly Asn His Gly Ser Cys 210 215 220Cys Pro Glu Met Asp Ile Trp Glu Ala Asn Lys Ile Ser Thr Ala Leu225 230 235 240Thr Pro His Pro Cys Asp Ser Ser Glu Gln Thr Met Cys Glu Gly Asn 245 250 255Asp Cys Gly Gly Thr Tyr Ser Asp Asp Arg Tyr Gly Gly Thr Cys Asp 260 265 270Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Asp Ser Phe 275 280 285Tyr Gly Pro Gly Lys Thr Ile Asp Thr Gly Ser Lys Met Thr Val Val 290 295 300Thr Gln Phe Ile Thr Asp Gly Ser Gly Ser Leu Ser Glu Ile Lys Arg305 310 315 320Tyr Tyr Val Gln Asn Gly Asn Val Ile Ala Asn Ala Asp Ser Asn Ile 325 330 335Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Asp Phe Cys Thr Ala Gln 340 345 350Lys Lys Ala Phe Gly Asp Glu Asp Ile Phe Ala Glu His Asn Gly Leu 355 360 365Ala Gly Ile Ser Asp Ala Met Ser Ser Met Val Leu Ile Leu Ser Leu 370 375 380Trp Asp Asp Tyr Tyr Ala Ser Met Glu Trp Leu Asp Ser Asp Tyr Pro385 390 395 400Glu Asn Ala Thr Ala Thr Asp Pro Gly Val Ala Arg Gly Thr Cys Asp 405 410 415Ser Glu Ser Gly Val Pro Ala Thr Val Glu Gly Ala His Pro Asp Ser 420 425 430Ser Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser Thr Phe 435 440 445Ser Ala Ser Ala 45064513PRTHypocrea ceramica 64Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp65 70 75 80Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100 105 110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120 125Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130 135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 165 170 175Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu225 230 235 240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 245 250 255Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr 260 265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 280 285Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr305 310 315 320Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu 340 345 350Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375 380Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr385 390 395 400Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 405 410 415Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 420 425 430Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr 450 455 460Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser465 470 475 480His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys 485 490 495Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 500 505 510Leu 65491PRTHypocrea jecorinaVARIANT(348)...(349)Xaa = Any Amino Acid 65Glu Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp1 5 10 15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val 20 25 30Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr 35 40 45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn 50 55 60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Thr Ser65 70 75 80Ala Tyr Ser Ser Glx Pro Gly Gly Gly Gly Gly Val Val Ile Phe Phe 85 90 95Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala Ser Asp Thr Thr Tyr 100 105 110Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser Phe Asp Val Asp Val 115 120 125Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met 130 135 140Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr Asn Thr Ala Gly Ala145 150 155 160Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys 165 170 175Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Glu Pro Ser Ser Asn 180 185 190Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser Cys Cys Ser Glu Met 195 200 205Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro His Pro 210 215 220Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly Asp Gly Cys Gly Gly225 230 235 240Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys 245 250 255Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser Phe Tyr Gly Pro Gly 260 265 270Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys Leu Thr Val Val Thr Gln 275 280 285Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr Tyr Val Gln Asp Gly Val 290 295 300Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly Ser Tyr Ser Gly Asn Glu305 310 315 320Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu Ala Glu Phe Gly Gly Ser 325 330 335Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln Phe Xaa Xaa Ala Thr Ser 340 345 350Gly Gly Met Val Leu Val Met Ser Leu Trp Asp Asp Tyr Tyr Ala Asn 355 360 365Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr 370 375 380Pro Gly Ala Val Arg Gly Ser Cys Ser Thr Ser Ser Gly Val Pro Ala385 390 395 400Gln Val Glu Ser Gln Ser Pro Asn Ala Lys Val Thr Phe Ser Asn Ile 405 410 415Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn Pro Ser Gly Gly Asn Pro 420 425 430Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr Thr Thr Thr Thr Ser Ser 435 440 445Ser Glx Pro Pro Pro Gly Ala His Arg Arg Tyr Gly Gln Cys Gly Gly 450 455 460Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln465 470 475 480Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Val 485 49066513PRTTrichoderma viride 66Met Tyr Gln Lys Leu Ala Leu Ile Ser Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala Gln Ser Ala Cys Thr Leu Gln Ala Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp65 70 75 80Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95Ser Thr Tyr Gly Val Thr Thr Ser Ala Asp Ser Leu Ser Ile Gly Phe 100 105 110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120 125Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130 135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Thr Lys Tyr Pro 165 170 175Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu225 230 235 240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 245 250 255Gly Asp Ser Cys Gly Gly Thr Tyr Ser Gly Asp Arg Tyr Gly Gly Thr 260 265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 280 285Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr305 310 315 320Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335Asp Tyr Ser Gly Asn Ser Leu Asp Asp Asp Tyr Cys Ala Ala Glu Glu 340 345 350Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375 380Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr385 390 395 400Asp Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Ser Ser Thr 405 410 415Ser Ser Gly Val Pro Ala Gln Leu Glu Ser Asn Ser Pro Asn Ala Lys 420 425 430Val Val Tyr Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr 450 455 460Pro Arg Pro Ala Thr Ser Thr Gly Ser Ser Pro Gly Pro Thr Gln Thr465 470 475 480His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ile Gly Pro Thr Val Cys 485 490 495Ala Ser Gly Ser Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 500 505 510Leu67514PRTTrichoderma viride 67Met Tyr Gln Lys Leu Ala Leu Ile Ser Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala Gln Ser Ala Cys Thr Leu Gln Ala Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp65 70 75 80Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95Ser Thr Tyr Gly Val Thr Thr Ser Ala Asp Ser Leu Ser Ile Gly Phe 100 105 110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120 125Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130 135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 165 170 175Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu225 230 235 240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Asp 245 250 255Gly Asp Ser Cys Gly Gly Thr Tyr Ser Gly Asp Arg Tyr Gly Gly Thr 260 265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 280 285Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr305 310 315 320Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335Asp Tyr Ser Gly Asn Ser Leu Asp Asp Asp Tyr Cys Ala Ala Glu Glu 340 345 350Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375 380Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr385 390 395 400Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 405 410 415Ser Ser Gly Val Pro Ala Gln Leu Glu Ser Asn Ser Pro Asn Ala Lys 420 425 430Val Val Tyr Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445Ser Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr 450 455 460Thr Arg Arg Pro Ala Thr Ser Thr Gly Ser Ser Pro Gly Pro Thr Gln465 470 475 480Thr His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 485 490 495Cys Ala Ser Gly Ser Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln 500 505 510Cys Leu68505PRTTrichoderma harzianum 68Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Ala Ala Arg1 5 10 15Ala Gln Gln Val Cys Thr Gln Gln Ala Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Thr Ala Ser Gly Cys Thr Pro Gln Gln Gly Ser Val

35 40 45Val Leu Asp Ala Asn Trp Arg Trp Thr His Asp Thr Lys Ser Thr Thr 50 55 60Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asp65 70 75 80Ala Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Asn Tyr Ser Gly 85 90 95Thr Tyr Gly Val Thr Thr Ser Gly Asp Ala Leu Thr Leu Gln Phe Val 100 105 110Thr Ala Ser Asn Val Gly Ser Arg Leu Tyr Leu Met Ala Asn Asp Ser 115 120 125Thr Tyr Gln Glu Phe Thr Leu Ser Gly Asn Glu Phe Ser Phe Asp Val 130 135 140Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val145 150 155 160Ser Met Asp Ala Asp Gly Gly Gln Ser Lys Tyr Pro Gly Asn Ala Ala 165 170 175Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp 180 185 190Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Glu Pro Ser 195 200 205Ser Asn Asn Ala Asn Thr Gly Val Gly Gly His Gly Ser Cys Cys Ser 210 215 220Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro225 230 235 240His Pro Cys Glu Thr Val Gly Gln Thr Met Cys Ser Gly Asp Ser Cys 245 250 255Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp 260 265 270Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr Ser Phe Tyr Gly 275 280 285Pro Gly Ser Ser Phe Ala Leu Asp Thr Thr Lys Lys Leu Thr Val Val 290 295 300Thr Gln Phe Ala Thr Asp Gly Ser Ile Ser Arg Tyr Tyr Val Gln Asn305 310 315 320Gly Val Lys Phe Gln Gln Pro Asn Ala Gln Val Gly Ser Tyr Ser Gly 325 330 335Asn Thr Ile Asn Thr Asp Tyr Cys Ala Ala Glu Gln Thr Ala Phe Gly 340 345 350Gly Thr Ser Phe Thr Asp Lys Gly Gly Leu Ala Gln Ile Asn Lys Ala 355 360 365Phe Gln Gly Gly Met Val Leu Val Met Ser Leu Trp Asp Asp Tyr Ala 370 375 380Val Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn Ala Thr Ala385 390 395 400Ser Thr Pro Gly Ala Lys Arg Gly Ser Cys Ser Thr Ser Ser Gly Val 405 410 415Pro Ala Gln Val Glu Ala Gln Ser Pro Asn Ser Lys Val Ile Tyr Ser 420 425 430Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr Gly Gly Asn Thr Gly Ser 435 440 445Asn Pro Pro Gly Thr Ser Thr Thr Arg Ala Pro Pro Ser Ser Thr Gly 450 455 460Ser Ser Pro Thr Ala Thr Gln Thr His Tyr Gly Gln Cys Gly Gly Thr465 470 475 480Gly Trp Thr Gly Pro Thr Arg Cys Ala Ser Gly Tyr Thr Cys Gln Val 485 490 495Leu Asn Pro Phe Tyr Ser Gln Cys Leu 500 50569506PRTAspergillus bisporus 69Met Phe Pro Arg Ser Ile Leu Leu Ala Leu Ser Leu Thr Ala Val Ala1 5 10 15Leu Gly Gln Gln Val Gly Thr Asn Met Ala Glu Asn His Pro Ser Leu 20 25 30Thr Trp Gln Arg Cys Thr Ser Ser Gly Cys Gln Asn Val Asn Gly Lys 35 40 45Val Thr Leu Asp Ala Asn Trp Arg Trp Thr His Arg Ile Asn Asp Phe 50 55 60Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser Ile Cys Pro Asp65 70 75 80Gly Val Thr Cys Ala Glu Asn Cys Ala Leu Asp Gly Ala Asp Tyr Ala 85 90 95Gly Thr Tyr Gly Val Thr Ser Ser Gly Thr Ala Leu Thr Leu Lys Phe 100 105 110Val Thr Glu Ser Gln Gln Lys Asn Ile Gly Ser Arg Leu Tyr Leu Met 115 120 125Ala Asp Asp Ser Asn Tyr Glu Ile Phe Asn Leu Leu Asn Lys Glu Phe 130 135 140Thr Phe Asp Val Asp Val Ser Lys Leu Pro Cys Gly Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Ser Glu Met Ala Ala Asp Gly Gly Met Ser Ser Thr Asn 165 170 175Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190Arg Asp Ile Lys Phe Ile Asp Gly Glu Ala Asn Ser Glu Gly Trp Glu 195 200 205Gly Ser Pro Asn Asp Val Asn Ala Gly Thr Gly Asn Phe Gly Ala Cys 210 215 220Cys Gly Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Ser Ala Tyr225 230 235 240Thr Pro His Pro Cys Arg Glu Pro Gly Leu Gln Arg Cys Glu Gly Asn 245 250 255Thr Cys Ser Val Asn Asp Arg Tyr Ala Thr Glu Cys Asp Pro Asp Gly 260 265 270Cys Asp Phe Asn Ser Phe Arg Met Gly Asp Lys Ser Phe Tyr Gly Pro 275 280 285Gly Met Thr Val Asp Thr Asn Gln Pro Ile Thr Val Val Thr Gln Phe 290 295 300Ile Thr Asp Asn Gly Ser Asp Asn Gly Asn Leu Gln Glu Ile Arg Arg305 310 315 320Ile Tyr Val Gln Asn Gly Gln Val Ile Gln Asn Ser Asn Val Asn Ile 325 330 335Pro Gly Ile Asp Ser Gly Asn Ser Ile Ser Ala Glu Phe Cys Asp Gln 340 345 350Ala Lys Glu Ala Phe Gly Asp Glu Arg Ser Phe Gln Asp Arg Gly Gly 355 360 365Leu Ser Gly Met Gly Ser Ala Leu Asp Arg Gly Met Val Leu Val Leu 370 375 380Ser Ile Trp Asp Asp His Ala Val Asn Met Leu Trp Leu Asp Ser Asp385 390 395 400Tyr Pro Leu Asp Ala Ser Pro Ser Gln Pro Gly Ile Ser Arg Gly Thr 405 410 415Cys Ser Arg Asp Ser Gly Lys Pro Glu Asp Val Glu Ala Asn Ala Gly 420 425 430Gly Val Gln Val Val Tyr Ser Asn Ile Lys Phe Gly Asp Ile Asn Ser 435 440 445Thr Phe Asn Asn Asn Gly Gly Gly Gly Gly Asn Pro Ser Pro Thr Thr 450 455 460Thr Arg Pro Asn Ser Pro Ala Gln Thr Met Trp Gly Gln Cys Gly Gly465 470 475 480Gln Gly Trp Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His 485 490 495Val Ile Asn Asp Phe Tyr Ser Gln Cys Phe 500 50570536PRTVolvariella volvacea 70Met Arg Ala Ser Leu Leu Ala Phe Ser Leu Asn Ser Ala Ala Gly Gln1 5 10 15Gln Ala Gly Thr Leu Gln Thr Lys Asn His Pro Ser Leu Thr Ser Gln 20 25 30Lys Cys Arg Gln Gly Gly Cys Pro Gln Val Asn Thr Thr Ile Val Leu 35 40 45Asp Ala Asn Trp Arg Trp Thr His Ser Thr Ser Gly Ser Thr Asn Cys 50 55 60Tyr Thr Gly Asn Thr Trp Gln Ala Thr Leu Cys Pro Asp Gly Lys Thr65 70 75 80Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr Thr Gly Thr Tyr 85 90 95Gly Val Thr Thr Ser Gly Asn Ser Leu Thr Leu Gln Phe Val Thr Gln 100 105 110Ser Asn Val Gly Ala Arg Leu Gly Tyr Leu Met Ala Asp Asp Thr Thr 115 120 125Tyr Gln Met Phe Asn Leu Leu Asn Gln Glu Phe Trp Phe Asp Val Asp 130 135 140Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Ser Ala145 150 155 160Met Ala Arg Thr Ala Ala Trp Met Pro Met Val Val Cys Ala Ser Thr 165 170 175Pro Leu Ile Ser Thr Arg Arg Ser Thr Ala Arg Leu Leu Arg Leu Pro 180 185 190Val Pro Pro Arg Ser Arg Tyr Gly Arg Gly Ile Cys Asp Ser Gln Cys 195 200 205Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Val Gln Gly Trp 210 215 220Gln Pro Ser Pro Asn Asp Thr Asn Ala Gly Thr Gly Asn Tyr Gly Ala225 230 235 240Cys Cys Asn Lys Met Asp Val Trp Glu Ala Asn Ser Ile Ser Thr Ala 245 250 255Tyr Thr Pro His Pro Cys Thr Gln Arg Gly Leu Val Arg Cys Ser Gly 260 265 270Thr Ala Cys Gly Gly Gly Ser Asn Arg Tyr Gly Ser Ile Cys Asp His 275 280 285Asp Gly Leu Gly Phe Gln Asn Leu Phe Gly Met Gly Arg Thr Arg Val 290 295 300Arg Ala Arg Val Gly Arg Val Lys Gln Phe Asn Arg Ser Ser Arg Val305 310 315 320Val Glu Pro Ile Ser Trp Thr Lys Gln Thr Thr Leu His Leu Gly Asn 325 330 335Leu Pro Trp Lys Ser Ala Asp Cys Asn Val Gln Asn Gly Arg Val Ile 340 345 350Gln Asn Ser Lys Val Asn Ile Pro Gly Met Pro Ser Thr Met Asp Ser 355 360 365Val Thr Thr Glu Phe Cys Asn Ala Gln Lys Thr Ala Phe Asn Asp Thr 370 375 380Phe Ser Phe Gln Gln Lys Gly Gly Met Ala Asn Met Ser Glu Ala Leu385 390 395 400Arg Arg Gly Met Val Leu Val Leu Ser Ile Trp Asp Asp His Ala Ala 405 410 415Asn Met Leu Trp Leu Asp Ser Ile Thr Ser Ala Ala Ala Cys Arg Ser 420 425 430Thr Pro Ser Glu Val His Ala Thr Pro Leu Arg Glu Ser Gln Ile Arg 435 440 445Ser Ser His Ser Arg Gln Thr Arg Tyr Val Thr Phe Thr Asn Ile Lys 450 455 460Phe Gly Pro Phe Asn Ser Thr Gly Thr Thr Tyr Thr Thr Gly Ser Val465 470 475 480Pro Thr Thr Ser Thr Ser Thr Gly Thr Thr Gly Ser Ser Thr Pro Pro 485 490 495Gln Pro Thr Gly Val Thr Val Pro Gln Gly Gln Cys Gly Gly Ile Gly 500 505 510Tyr Thr Gly Pro Thr Thr Cys Ala Ser Pro Thr Thr Cys His Val Leu 515 520 525Asn Pro Tyr Tyr Ser Gln Cys Tyr 530 53571463PRTTrichoderma longibrachiatum 71Met Ala Pro Ser Ala Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile1 5 10 15Gly Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25 30His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Thr Ser Gly Gly Cys Val 35 40 45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55 60Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr65 70 75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Tyr Ile Glu Gly 85 90 95Val Asp Tyr Ala Ala Ser Gly Val Thr Ala Ser Gly Ser Thr Leu Thr 100 105 110Leu Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120 125Pro Arg Leu Tyr Leu Leu Gly Pro Asp Gly Glu Tyr Val Met Leu Lys 130 135 140Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145 150 155 160Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170 175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185 190Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195 200 205Thr Ser Gly Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210 215 220Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala225 230 235 240Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Pro 245 250 255Asn Tyr Phe Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285Val Ser Ile Thr Arg Lys Tyr Arg Gln Asn Gly Val Asp Ile Pro Ser 290 295 300Ala Lys Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala305 310 315 320Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val 325 330 335Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345 350Asp Ser Gly Arg Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365Asn Ile Leu Ala Asn Asn Pro Gly Thr His Val Val Tyr Ser Asn Ile 370 375 380Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Gly Gly Asn Pro385 390 395 400Pro Pro Pro Pro Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg 405 410 415Arg Ser Ser Thr Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp 420 425 430Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys Lys Thr Cys Thr Ser 435 440 445Gly Thr Thr Cys Gln Tyr Gly Asn Asp Tyr Tyr Ser Gln Cys Leu 450 455 46072459PRTHypocrea jecorina 72Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile1 5 10 15Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25 30His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40 45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55 60Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr65 70 75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly 85 90 95Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr 100 105 110Met Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120 125Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys 130 135 140Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145 150 155 160Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170 175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185 190Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195 200 205Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210 215 220Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala225 230 235 240Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245 250 255Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285Val Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295 300Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala305 310 315 320Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val 325 330 335Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345 350Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365Asn Ile Leu Ala Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370 375 380Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro385 390 395 400Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr 405 410 415Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420 425 430Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys 435 440 445Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450 45573416PRTAspergillus oryzae 73Met Ile Trp Thr Leu Ala Pro Phe Val Ala Leu Leu Pro Leu Val Thr1 5 10 15Ala Gln Gln Val Gly Thr Thr Ala Asp Ala His Pro Arg Leu Thr Thr

20 25 30Tyr Lys Cys Thr Ser Gln Asn Gly Cys Thr Arg Gln Asn Thr Ser Leu 35 40 45Val Leu Asp Ala Ala Thr His Phe Ile His Lys Lys Gly Thr Gln Thr 50 55 60Ser Cys Thr Asn Ser Asn Gly Leu Asp Thr Ala Ile Cys Pro Asp Lys65 70 75 80Gln Thr Cys Ala Asp Asn Cys Val Val Asp Gly Ile Thr Asp Tyr Ala 85 90 95Ser Tyr Gly Val Gln Thr Lys Asn Asp Thr Leu Thr Leu Gln Gln Tyr 100 105 110Leu Gln Thr Gly Asn Ala Thr Lys Ser Leu Ser Pro Arg Val Tyr Leu 115 120 125Leu Ala Glu Asp Gly Glu Asn Tyr Ser Met Leu Lys Leu Leu Asn Gln 130 135 140Glu Phe Thr Phe Asp Val Asp Ala Ser Thr Leu Val Cys Gly Met Asn145 150 155 160Gly Ala Leu Tyr Leu Ser Glu Met Glu Ala Ser Gly Gly Lys Ser Ser 165 170 175Leu Asn Gln Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ala Gln 180 185 190Cys Tyr Thr Thr Pro Trp Ile Asn Gly Glu Gly Asn Thr Glu Ser Val 195 200 205Gly Ser Cys Cys Gln Glu Met Asp Ile Trp Glu Ala Asn Ala Arg Ala 210 215 220Thr Gly Leu Thr Pro His Pro Cys Asn Thr Thr Gly Leu Tyr Glu Cys225 230 235 240Ser Gly Ser Gly Cys Gly Asp Ser Gly Val Cys Asp Lys Ala Gly Cys 245 250 255Gly Phe Asn Pro Tyr Gly Leu Gly Ala Lys Asp Tyr Tyr Gly Tyr Gly 260 265 270Leu Lys Val Asn Thr Asn Glu Thr Phe Thr Val Val Thr Gln Phe Leu 275 280 285Thr Asn Asp Asn Thr Thr Ser Gly Gln Leu Ser Glu Ile Arg Arg Leu 290 295 300Tyr Ile Gln Asn Gly Gln Val Ile Gln Asn Ala Ala Val Thr Ser Gly305 310 315 320Gly Lys Thr Val Asp Ser Ile Thr Lys Asp Phe Cys Ser Gly Glu Gly 325 330 335Ser Ala Phe Asn Arg Leu Gly Gly Leu Glu Glu Met Gly His Ala Leu 340 345 350Gly Arg Gly Met Val Leu Ala Leu Ser Ile Trp Asn Asp Ala Gly Ser 355 360 365Phe Met Gln Trp Leu Asp Gly Gly Ser Ala Gly Pro Cys Asn Ala Thr 370 375 380Glu Gly Asn Pro Ala Leu Ile Glu Lys Leu Tyr Pro Asp Thr His Val385 390 395 400Lys Phe Ser Lys Ile Arg Trp Gly Asp Ile Gly Ser Thr Tyr Arg His 405 410 41574381PRTArtificial Sequenceconsensus sequence 74Met Phe Ile Leu Met Val Ala Ala Gln Gln Gly Thr Thr Ala Glu His1 5 10 15Pro Leu Thr Trp Gln Lys Cys Thr Gly Cys Thr Gly Ser Val Val Leu 20 25 30Asp Ala Asn Trp Arg Trp Ile His Thr Gly Tyr Thr Asn Cys Tyr Thr 35 40 45Gly Asn Trp Asp Ser Thr Leu Cys Pro Asp Thr Cys Ala Asn Cys Ala 50 55 60Leu Asp Gly Ala Asp Tyr Ser Gly Thr Tyr Gly Ile Thr Thr Ser Gly65 70 75 80Ser Leu Ser Leu Phe Val Thr Gly Ser Asn Val Gly Ser Arg Val Tyr 85 90 95Leu Met Ala Asp Asp Thr Tyr Gln Met Phe Leu Leu Asn Asn Glu Phe 100 105 110Thr Phe Asp Val Asp Met Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala 115 120 125Leu Tyr Phe Val Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn 130 135 140Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro145 150 155 160Arg Asp Leu Lys Phe Ile Asn Gly Ala Asn Val Glu Gly Trp Ser Ser 165 170 175Asn Gly Gly Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn 180 185 190Ser Ile Ala Ala Phe Thr Pro His Pro Cys Thr Thr Gly Gln Thr Cys 195 200 205Gly Asp Cys Gly Gly Thr Tyr Ser Asp Arg Tyr Gly Cys Asp Asp Gly 210 215 220Cys Asp Phe Asn Tyr Arg Met Gly Asn Ser Phe Tyr Gly Gly Thr Val225 230 235 240Asp Thr Thr Lys Lys Phe Thr Val Val Thr Gln Phe Val Thr Ser Gly 245 250 255Leu Glu Ile Arg Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Asn Ile 260 265 270Pro Gly Val Gly Asn Ser Ile Thr Asp Glu Phe Cys Gln Lys Phe Gly 275 280 285Asp Ser Phe Gly Gly Leu Gln Met Gly Ala Leu Gly Met Val Leu Val 290 295 300Met Ser Ile Trp Asp Asp His Ala Ala Asn Met Leu Trp Leu Asp Ser305 310 315 320Tyr Pro Thr Ser Pro Gly Arg Gly Ser Cys Thr Thr Ser Gly Val Pro 325 330 335Ala Val Glu Gln Pro Asn Val Val Phe Ser Asn Ile Lys Phe Gly Pro 340 345 350Ile Gly Ser Thr Tyr Gly Ser Ser Phe Gly Gln Cys Gly Gly Gly Tyr 355 360 365Thr Gly Thr Cys Ser Thr Cys Asn Tyr Tyr Ser Gln Cys 370 375 3807521DNAArtificial Sequencesynthetic oligonucleotide 75ggtttggatc cggtcaccag g 217627DNAArtificial Sequencesynthetic oligonucleotide 76cgcgcctggt gaccggatcc aaaccgc 2777353PRTArtificial Sequenceconsensus sequence 77Thr Pro Gly Thr Thr Lys Glu Val His Pro Lys Leu Thr Thr Tyr Arg1 5 10 15Cys Thr Lys Ala Gly Gly Cys Lys Gln Thr Asn Ser Ile Val Leu Asp 20 25 30Ala Asn Trp Trp Ile His Asn Cys Gly Cys Gly Asp Trp Gly Gln Pro 35 40 45Asn Ser Thr Leu Cys Pro Asp Glu Ser Cys Ala Lys Asn Cys Ile Leu 50 55 60Glu Gly Met Ala Tyr Ala Asn Tyr Gly Val Thr Thr Ser Gly Asn Ser65 70 75 80Leu Arg Leu Gln Gln Leu Ile Pro Ser Asn Arg Leu Val Ser Pro Arg 85 90 95Val Tyr Leu Leu Asp Thr Lys Lys Tyr Glu Met Leu His Leu Thr Gly 100 105 110Asn Glu Phe Ser Phe Asp Val Asp Met Ser Lys Leu Pro Cys Gly Met 115 120 125Asn Gly Ala Leu Tyr Leu Ser Glu Met Asp Asp Gly Gly Lys Ser Arg 130 135 140Tyr Asn Thr Ala Gly Ala Tyr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln145 150 155 160Cys Pro Val Thr Pro Phe Ile Asn Gly Val Gly Asn Ile Glu Gly Gln 165 170 175Gly Ser Cys Cys Asn Glu Met Asp Ile Trp Asx Ala Asn Ser Arg Ala 180 185 190Thr Leu Pro His Pro Cys Thr Lys Gly Leu Tyr Leu Cys Glu Gly Asp 195 200 205Glu Cys Gly Phe Gly Ile Cys Asp Lys Ala Gly Cys Gly Trp Asn Pro 210 215 220Tyr Arg Ile Val Thr Phe Tyr Gly Gly Phe Val Asp Thr Thr Lys Lys225 230 235 240Phe Thr Val Val Thr Gln Phe Val Asn Lys Gly Leu Ile Ile His Arg 245 250 255Phe Tyr Val Gln Gly Val Ile Glu Ser Ala Asn Asn Gly Pro Gly Asn 260 265 270Ile Asn Asp Glu Tyr Cys Ala Thr Gly Ala Ser Tyr Glu Leu Gly Gly 275 280 285Gln Met Gly Lys Ala Leu Ser Arg Gly Asn Val Leu Met Ser Ile Trp 290 295 300Trp Asp Gln Gly Gly Asn Met Trp Leu Asp Ser Gly Val Ala Gly Pro305 310 315 320Cys Ser Thr Thr Glu Gly Pro Ser Asn Ile Val Val Gln Pro Asn Pro 325 330 335Glu Val Thr Phe Ser Asn Ile Arg Trp Gly Glu Ile Gly Ser Thr Ser 340 345 350Gln

Claims

1-23. (canceled)

24. A nucleic acid encoding a Hypocrea jecorina CBH I variant consisting essentially of an amino acid sequence having at least 96.4% sequence identity to the amino acid sequence of SEQ ID NO:2, having CBH I cellulase activity, and comprising one or more amino acid substitutions selected from the group consisting of Q17L, G22D, T41I, N49S, S57N, A68T, A77D, S92T, N103I, A112E, S113(T/N/D), E193V, S196T, M213I, P227(L/T/A), T246(C/A), D249K, Y252(A/Q), T255P, D257E, D259W, S278P, S279N, K286M, L288F, E295K, T296P, S297T, N301(R/K), E325K, T332(K/Y/H), F338Y, S342Y, F352L, T356L, Y371C, T380G, Y381D, V393G, S398T, V403D, S411F, G430F, G440R, T462I, T484S, Q487L, and P491L.

25. The nucleic acid of claim 64, wherein the CBH1 variant comprises one or more amino acid substitutions selected from the group consisting of N49S, A68T, A77D, S92T, S113(N/D), P227(A/L/T), D249K, T255P, D257E, S279N, L288F, E295K, S297T, N301(R/K), T332(K/Y/H), F338Y, T356L, V393G, and G430F.

26. A vector comprising a nucleic acid of claim 24.

27. A host cell transformed with the vector of claim 26.

28. A method of producing a CBH I variant comprising the steps of: (a) culturing the host cell according to claim 27 in a suitable culture medium under suitable conditions to produce a CBH I variant; and (b) obtaining said produced CBH I variant.

29. A recombinant filamentous fungal cell engineered to be capable of expressing a CBH I variant encoded by the nucleic acid of claim 24.

30. A method of producing a CBH I variant comprising the steps of: (a) culturing the recombinant filamentous fungal cell according to claim 29 in a suitable culture medium under suitable conditions to express a CBH I variant; and (b) obtaining said CBH I variant.

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