Patent application title: O-Mannosyltransferase Deficient Filamentous Fungal Cells and Methods of Use Thereof
Inventors:
Jari Natunen (Vantaa, FI)
Jukka Hiltunen (Helsinki, FI)
Jukka Hiltunen (Helsinki, FI)
Anne Huuskonen (Helsinki, FI)
Markku Saloheimo (Helsinki, FI)
Markku Saloheimo (Helsinki, FI)
Christian Ostermeier (Basel, CH)
Benjamin Patrick Sommer (Basel, CH)
Ramon Wahl (Basel, CH)
IPC8 Class: AC12N910FI
USPC Class:
5303873
Class name: Globulins immunoglobulin, antibody, or fragment thereof, other than immunoglobulin antibody, or fragment thereof that is conjugated or adsorbed chimeric, mutated, or recombined hybrid (e.g., bifunctional, bispecific, rodent-human chimeric, single chain, rfv, immunoglobulin fusion protein, etc.)
Publication date: 2016-12-29
Patent application number: 20160376570
Abstract:
The present disclosure relates to compositions and methods useful for the
production of heterologous proteins with reduced O-mannosylation in
filamentous fungal cells, such as Trichoderma cells. More specifically,
the invention provides a PMT-deficient filamentous fungal cell comprising
a) at least a first mutation that reduces an endogenous protease activity
compared to a parental filamentous fungal cell which does not have said
first mutation, and, b) at least a second mutation in a PMT gene that
reduces endogenous O-mannosyltransferase activity compared to a parental
filamentous fungal cell which does not have said second mutation, wherein
said filamentous fungal cell is selected from the group consisting of
Trichoderma, Neurospora, Myceliophthora or Chrysosporium cell.Claims:
1. A PMT-deficient filamentous fungal cell comprising: a) a first
mutation that reduces or eliminates an endogenous protease activity
compared to a parental filamentous fungal cell which does not have said
first mutation, and b) a second mutation in a PMT gene that reduces
endogenous O-mannosyltransferase activity compared to a parental
filamentous fungal cell which does not have said second mutation, wherein
said filamentous fungal cell is selected from the group consisting of
Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.
2. The PMT-deficient filamentous fungal cell of claim 1, wherein said second mutation that reduces the endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding an endogenous protein O-mannosyltransferase activity.
3. The PMT-deficient filamentous fungal cell of claim 1, wherein said second mutation in a PMT gene is a mutation in either: a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1, b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ NO:2, said polypeptide having O-mannosyltransferase activity.
4. The PMT-deficient filamentous fungal cell of claim 1, wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation.
5. The PMT-deficient filamentous fungal cell of claim 4, further comprising a first polynucleotide encoding a N-acetylglucosaminyltransferase I catalytic domain and a second polynucleotide encoding a N-acetylglucosaminyltransferase IT catalytic domain.
6. The PMT-deficient filamentous fungal cell of claim 1-5, further comprising one or more polynucleotides encoding a polypeptide selected from the group consisting of: a) .alpha.1,2 mannosidase; b) N-acetylglucosaminyltransferase I catalytic domain; c) .alpha. mannosidase d) N-acetylglucosaminyltransferase II catalytic domain; e) .beta.1,4 galactosyltransferase; and, f) fucosyltransferase.
7. The PMT-deficient filamentous fungal cell of claim 1, wherein said cell is a Trichoderma cell comprising a mutation that reduces or eliminates the protein O-mannosyltransferase activity of Trichoderma pmt1.
8. The PMT-deficient filamentous fungal cell of claim 1, wherein said cell is a Trichoderma cell, for example Trichoderma reesei, and said cell comprises mutations that reduce or eliminate the activity of: a) the three endogenous proteases pep1, tsp1 and slp1; b) the three endogenous proteases gap1, slp1 and pep1; c) the three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1 and gap2; d) three to six proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2; or e) seven to ten proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.
9. A method for producing a protein having reduced O-mannosylation, comprising: a) providing a PMT-deficient filamentous fungal cell having a mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity in comparison to a parental strain which does not have such mutation, and further comprising a polynucleotide encoding a protein with serine or threonine residue, b) culturing said PMT-deficient filamentous fungal cell to produce said protein having reduced O-mannosylation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora or Chrysosporium cell.
10. The method according to claim 9, wherein said mutation in a PMT gene is a mutation in either: a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1, b) a functional homologous gene of PMT1 gene, which gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.
11. The method according to claim 9, wherein said filamentous fungal cell expresses functional endogenous chaperone protein, such as Protein Disulphide Isomerase (PDI).
12. The method according to any one of claim 9, wherein said PMT-deficient filamentous fungal cell is selected from the cells as defined in any one of claims 1-8.
13. The method of claim 9, wherein said produced protein is a heterologous mammalian protein selected from the group consisting of a) an immunoglubulin, such as IgG, b) a light chain or heavy chain of an immunoglobulin, c) a heavy chain or a light chain of an antibody, d) a single chain antibody, e) a camelid antibody, f) a monomeric or multimeric single domain antibody, g) a FAb-fragment, a FAb2-fragment, and, h) their antigen-binding fragments.
14. A method for producing an antibody having reduced O-mannosylation, comprising: a) providing a PMT-deficient filamentous fungal cell having: i. a mutation that reduces endogenous protein O-mannosyltransferase activity as compared to parental strain which does not have such mutation, and, ii. a polynucleotide encoding a light chain antibody and a polynucleotide encoding a heavy chain antibody, b) culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.
15. A protein or antibody composition obtainable by the method of claim 9.
16. The protein or antibody composition according to claim 15, wherein said protein or antibody comprises, as a major glycoform, either, a) Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.4Glc- NAc (Man5 glycoform); b) Man.alpha.6(Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc (Man3 glycoform); c) hybrid or complex type N-glycans, such as the glycoforms selected from the subgroup consisting of GlcNAcMan3, G0, hybrid glycan GlcNAcMan5, and galactosylated derivatives, such as GalGlcNAcMan3, G1, G2; and, GalGlcNAcMan5 glycoforms.
17. The PMT-deficient filamentous fungal cell of claim 2, wherein said second mutation in a PMT gene is a mutation in either: a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1, b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.
18. The PMT-deficient filamentous fungal cell of claim 2, wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation.
19. The PMT-deficient filamentous fungal cell of claim 3, wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation.
20. A protein or antibody composition obtainable by the method of claim 14.
Description:
FIELD OF THE INVENTION
[0001] The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells.
BACKGROUND
[0002] Posttranslational modification of eukaryotic proteins, particularly therapeutic proteins such as immunoglobulins, is often necessary for proper protein folding and function. Because standard prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in production of these therapeutic proteins. Even where eukaryotic proteins do not have posttranslational modifications, prokaryotic expression systems often lack necessary chaperone proteins required for proper folding. Yeast and fungi are attractive options for expressing proteins as they can be easily grown at a large scale in simple media, which allows low production costs, and yeast and fungi have posttranslational machinery and chaperones that perform similar functions as found in mammalian cells. Moreover, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells as well as more complex eukaryotic cells such as mammalian or insect cells (De Pourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31).
[0003] However, posttranslational modifications occurring in yeast and fungi may still be a concern for the production of recombinant therapeutic protein. In particular, O-mannosylation is one of the biggest hurdles to overcome in the production of biopharmaceuticals for human applications in fungi. More specifically, yeasts like Pichia pastoris and Saccharomyces cerevisiae tend to hyper-mannosylate heterologously expressed biopharmaceuticals, thereby triggering adverse effects when applied to humans.
[0004] O-mannosylation to Serine and Threonine residues includes in mammals GalNAc based oligosaccharides or GlcNAc/N-acetyllactosamine comprising O-linked mannose glycans. In fungi O-mannosylation occurs as hexose monomers or oligomers. In yeasts, there are typically several protein(/polypeptide) O-mannosyltransferases, which often function as complexes. Part of the knock-outs are harmful, at least for cell structures and stability and not all yeast knock-outs or combinations are tolerated (for a review, see Goto 2007, Biosci. Biotechnol. Biochem. 71(6), 1415-1427).
[0005] There have been reports of knock-outs of yeast O-mannosyltransferase genes, aiming to reduce the O-mannosylation levels, and even multiple knock-out mutants involving two or three pmt genes in S. cerevisiae (WO/1994/004687). Pmt1 or pmt2 knock-out of S. cerevisiae reduced the level of O-mannosylation of antifreeze glycoprotein III to about 30% of the proteins and the residual mannosylated protein contains numerous mannose residues per protein, apparently also oligosaccharides (WO/2004/057007).
[0006] WO/2010/034708 reports no significant level of O-mannosylation of recombinant hydrophobin Trichoderma protein when expressed in pmt1 knock-out of S. cerevisiae host cell. Such O-mannosylation appears to be artificial yeast glycosylation of the original non-mannosylated filamentous fungal protein.
[0007] WO/2010/128143 further reports single chain antibody-albumin fusion construct in yeast S. cerevisiae pmt1 and/or pmt4 knock-out strains.
[0008] Pmt1, pmt2, and pmt3 single gene knock-outs, double, and triple knock-outs of
[0009] Aspergillus species (Aspergillus nidulans, Aspergillus fumigatus, and/or Aspergillus awamori) are described in Goto et al, 2009 (Eukaryotic cell 2009, 8(10):1465); Mouyna et al, 2010 (Molecular Microbiology 2010, 76(5), 1205-1221); Zhou et al, 2007 (Eukaryotic cell 2007, 6(12):2260); Oka et al, 2004 (Microbiology 2004, 150, 1973-1982); Kriangkripipat et al, 2009; Fang et al, 2010 (Glycobiology, 2010, vol. 20 pp 542-552); and Oka et al, 2005 (Microbiology 2005, 151, 3657-3667).
[0010] Despite numerous reports on knock out of pmt homologues in filamentous fungi, there is no description of a filamentous fungal cell with reduced O-mannosylation and useful as a host cell for the production of recombinant glycoprotein.
[0011] In particular, Gorka-Niec et al (2008, Acta Biochimica Polonica, Vol. 55 No 2/2008, 251-259) reported the deletion of pmt1 gene in Trichoderma reesei. PMT1 protein showed the highest identity to Pmt4p of S. cerevisiae (51%) but functionally complement pmt2.DELTA. S. cerevisiae mutant (Gorka-Niec et al, 2007, Biochimica et Biophysica Acta 1770, 2007, 774-780). However, the authors reported that disruption of the pmt1 gene caused a decrease of protein secretion but did not alter O- and N-glycosylation of secreted protein. Zakrzewska et al (Curr Genet 2003 43: 11-16) further reported that Trichoderma reesei pmt1 gene did not functionally complement pmt4.DELTA. S. cerevisiae mutant.
[0012] In fact, deletions of the PMT genes in yeasts or filamentous fungi appears to either result in no phenotype at all or lethality or severely impaired vital functions of the cells, which would not be suitable for recombinant production of heterologous proteins, especially mammalian glycoproteins. For this reason, alternative methods such as the use of pmt inhibitors have been proposed as an alternative to pmt knock out strains (WO2009/143041).
[0013] Thus, a need remains for improved filamentous fungal cells, such as Trichoderma fungus cells, that can stably produce heterologous proteins with no or reduced O-mannosylation, such as immunoglobulins, preferably at high levels of expression.
SUMMARY
[0014] The present invention relates to improved methods for producing proteins with no or reduced O-mannosylation in filamentous fungal expression systems, and more specifically, glycoproteins, such as antibodies or related immunoglobulins or fusion proteins which may be O-mannosylated when produced in filamentous fungal expression systems.
[0015] The present invention is based in part on the surprising discovery that filamentous fungal cells, such as Trichoderma cells, can be genetically modified to reduce or suppress O-mannosylation activity, without adversely affecting viability and yield of produced glycoproteins.
[0016] Accordingly, in a first aspect, the invention relates to a PMT-deficient filamentous fungal cell comprising
[0017] a) a first mutation that reduces or eliminates an endogenous protease activity compared to a parental filamentous fungal cell which does not have said first mutation, and,
[0018] b) a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.
[0019] In one embodiment, said PMT-deficient cell further expresses a heterologous protein containing serine and/or threonine residues. The expressed heterologous protein with serine and/or threonine residues has reduced O-mannosylation due to said mutation in said PMT gene. For example, the O-mannosylation level of the heterologous protein expressed in a PMT-deficient cell of the invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% lower as compared to the O-mannosylation level of the heterologous protein when expressed in the parental filamentous fungal cell which does not have said second PMT-deficient mutation.
[0020] In another embodiment, said second mutation that reduces endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding an endogenous protein O-mannosyltransferase activity.
[0021] In another embodiment, said second PMT-deficient mutation in a PMT gene may be a mutation (such as a deletion or disruption) in either:
[0022] a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,
[0023] b) a functional homologous gene of PMT1 gene, which functional gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or,
[0024] c) a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.
[0025] In another embodiment that may be combined with the precedent embodiments, said PMT-deficient cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation. In a specific embodiment, said PMT-deficient cell further comprises a first polynucleotide encoding N-acetylglucosaminyltransferase I catalytic domain and a second polynucleotide encoding N-acetylglucosaminyltransferase II catalytic domain.
[0026] In another embodiment that may be combined with the preceding embodiments, said PMT-deficient cell further comprises one or more polynucleotides encoding a polypeptide selected from the group consisting of:
[0027] a) .alpha.1,2 mannosidase,
[0028] b) N-acetylglucosaminyltransferase I catalytic domain,
[0029] c) .alpha. mannosidase II, and
[0030] d) N-acetylglucosaminyltransferase II catalytic domain.
[0031] In another embodiment that may be combined with the preceding embodiments, said PMT-deficient cell further comprises one or more polynucleotides encoding a .beta.1,4 galactosyltransferase and/or a fucosyltransferase.
[0032] In one specific embodiment, said PMT-deficient cell is a Trichoderma cell comprising at least a mutation that reduces or eliminates the protein O-mannosyltransferase activity of Trichoderma pmt1, and, optionally, further comprising mutations in at least one or more other PMT genes that reduces or eliminates the protein O-mannosyltransferase activity selected from the group consisting of pmt2 and pmt3.
[0033] In one embodiment that may be combined with the preceding embodiments, the PMT deficient cells comprise mutations that reduce or eliminate the activity of at least two, or at least three endogenous proteases. Typically, said cell may be a Trichoderma cell and may comprise mutations that reduce or eliminate the activity of
[0034] a) the three endogenous proteases pep1, tsp1 and slp1,
[0035] b) the three endogenous proteases gap1, slp1 and pep1,
[0036] c) three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1 and gap2,
[0037] d) three to six proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2, or,
[0038] e) seven to ten proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.
[0039] In one embodiment that may be combined with the precedent embodiments, the filamentous fungal cell of the invention does not comprise a deletion or disruption of an endogenous gene encoding a chaperone protein. In particular, said filamentous fungal cell of the invention expresses functional endogenous chaperone protein Protein Disulphide Isomerase (PDI).
[0040] In another aspect, the invention relates to a method for producing a protein having reduced O-mannosylation, comprising:
[0041] a) providing a PMT-deficient filamentous fungal cell, having a mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity as compared to parental strain which does not have such mutation, and further comprising a polynucleotide encoding a protein with serine or threonine residue,
[0042] b) culturing said PMT-deficient filamentous fungal cell to produce said protein with reduced O-mannosylation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.
[0043] According to one specific embodiment of the method, said mutation in a PMT gene is a mutation, such as a deletion or disruption, in either:
[0044] a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,
[0045] b) a functional homologous gene of PMT1 gene, which gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or,
[0046] c) a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.
[0047] In another embodiment of the method, said PMT-deficient cell is a Trichoderma reesei cell and said mutation is a deletion or a disruption of T. reesei PMT1 gene.
[0048] In other embodiments of the method, said PMT-deficient cell is a PMT-deficient cell of the invention as described above.
[0049] In a specific embodiment, said polynucleotide encoding a protein is a recombinant polynucleotide encoding a heterologous protein. Typically, said heterologous protein may be a mammalian protein selected from the group consisting of
[0050] a) an immunoglubulin, such as IgG,
[0051] b) a light chain or heavy chain of an immunoglobulin,
[0052] c) a heavy chain or a light chain of an antibody,
[0053] d) a single chain antibody,
[0054] e) a camelid antibody,
[0055] f) a monomeric or multimeric single domain antibody,
[0056] g) a FAb-fragment, a FAb2-fragment, and,
[0057] h) their antigen-binding fragments.
[0058] In one embodiment of the method, that may be combined with the preceding embodiments, said polynucleotide encoding said protein further comprises a polynucleotide encoding CBH1 catalytic domain and linker as a carrier protein and/or cbh1 promoter.
[0059] In another embodiment, said polynucleotide encodes a protein with serine or threonine, which may be O-mannosylated in a PMT functional parental strain, and further comprising at least one N-glycan.
[0060] The invention also relates to a method for producing an antibody having reduced 0-mannosylation, comprising:
[0061] a) providing a PMT-deficient filamentous fungal cell having
[0062] i. a mutation that reduces endogenous protein O-mannosyltransferase activity as compared to parental strain which does not have such mutation and
[0063] ii. a polynucleotide encoding a light chain antibody and a polynucleotide encoding a heavy chain antibody,
[0064] b) culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation,
[0065] wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.
[0066] In a specific method for producing antibody, said PMT-deficient cell is a Trichoderma reesei cell and said mutation is a deletion or a disruption of T. reesei PMT1 gene.
[0067] In one embodiment of the method for producing antibody, at least 70%, 80%, 90%, 95%, or 100% of the produced antibody is not O-mannosylated.
[0068] The invention also relates to the protein composition or antibody composition obtainable or obtained by the methods of the invention as described above. In one embodiment, at least 70%, 80%, 90%, 95%, or 100% of the antibodies as obtained or obtainable the methods of the invention are not O-mannosylated.
[0069] In one specific embodiment, such protein (e.g. a glycoprotein) or antibody composition with reduced O-mannosylation comprises, as a major glycoform, either,
[0070] Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.4Glc- NAc (Man5 glycoform);
[0071] Man.alpha.6(Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc (Man3 glycoform);
[0072] hybrid or complex type N-glycans such as glycoforms selected from the subgroup consisting of GlcNAcMan3, G0, hybrid glycan, or GlcNAcMan5, or galactosylated derivatives, such as GalGlcNAcMan3, G1, G2; or, GalGlcNAcMan5 glycoform.
[0073] In one specific embodiment, when the core of the glycan consists of Man3, then the composition essentially lacks Man5 glycoforms.
[0074] In an embodiment that may be combined with one or more of the preceding embodiments less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/or O-glycans of the protein composition comprises Neu5Gc and/or Gal.alpha.- structure. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/or O-glycans of the antibody composition comprises Neu5Gc and/or Gal.alpha.- structure.
[0075] In an embodiment that may be combined with one or more of the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the glycoprotein composition comprises core fucose structures. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the antibody composition comprises core fucose structures.
[0076] In an embodiment that may be combined with one or more of the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of N-glycan of the glycoprotein composition comprises terminal galactose epitopes Gal.beta.3/4GlcNAc. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the antibody composition comprises terminal galactose epitopes Gal.beta.3/4GlcNAc.
[0077] In an embodiment that may be combined with one or more of the preceding embodiments, less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the glycoprotein composition comprises glycation structures. In an embodiment that may be combined with the preceding embodiments, less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the antibody composition comprises glycation structures.
[0078] In another embodiment that may be combined with one or more of the preceding embodiments, the glycoprotein composition, such as an antibody is devoid of one, two, three, four, five, or six of the structures selected from the group of Neu5Gc, terminal Gal.alpha.3Gal.beta.4GlcNAc, terminal Gal.beta.4GlcNAc, terminal Gal.beta.3GlcNAc, core linked fucose and glycation structures.
[0079] The invention also relates to a method of reducing O-mannosylation level of a recombinant glycoprotein composition produced in a filamentous fungal cell, for example, Trichoderma cell, typically, Trichoderma reesei, said method consisting of using a filamentous fungal cell having a mutation in a PMT gene wherein said PMT gene is either:
[0080] i. PMT1 gene comprising the polynucleotide of SEQ ID NO:1,
[0081] ii. a functional homologous gene of PMT1 gene, which gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or,
[0082] iii. a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.
DESCRIPTION OF THE FIGURES
[0083] FIG. 1 depicts results for Southern analyses of Trichoderma reesei pmt1 deletion strains expressing antibody MAB01. A) A 5.7 kb signal is expected from parental strains M124 and M304 with pmt1 ORF probe after SpeI+XbaI digestion. No signal is expected from pure pmt1 deletion strains. B) A 3.5 kb signal is expected for pmt1 5'flank probe from deletion strains after SpeI+AscI digestion. C) A 1.7 kb signal is expected for pmt1 3'flank probe from deletion strains after AscI+XbaI digestions. AscI does not cut intact pmt1 locus in close distance, therefore signals of over 16 kb (B) and 10 kb (C) are expected from parental strains M124 or M304. A 4.1 kb signal is expected from PmeI digested plasmid pTTv185 used as a control in hybridisations with both flank probes (B, C).
[0084] FIG. 2 depicts a spectra of light chain of flask cultured parental T. reesei strain M317 (pyr4.sup.- of M304) (A) and .DELTA.pmt1 disruptant clone 26-8A (B), day 7.
[0085] FIG. 3 depicts results for Western analyses of Trichoderma reesei pmt1 deletion strain M403 from fed-batch fermentation. Upper panel: MAB01 light chain, lower panel: MAB01 heavy chain. 0.1 .mu.l of supernatant was loaded on each lane.
[0086] FIG. 4 depicts a spectrum of light chain of fermenter cultured T. reesei strain M403 (pmt1 deletion strain of MAB01 antibody producing strain, clone 26-8A), day 7.
[0087] FIG. 5 depicts a phylogeny of PMTs of selected filamentous fungi.
[0088] FIG. 6 depicts a partial sequence alignment of the results of the PMT BLAST searches.
DETAILED DESCRIPTION
Definitions
[0089] As used herein, an "expression system" or a "host cell" refers to the cell that is genetically modified to enable the transcription, translation and proper folding of a polypeptide or a protein of interest, typically of mammalian protein.
[0090] The term "polynucleotide" or "oligonucleotide" or "nucleic acid" as used herein typically refers to a polymer of at least two nucleotides joined together by a phosphodiester bond and may consist of either ribonucleotides or deoxynucleotides or their derivatives that can be introduced into a host cell for genetic modification of such host cell. For example, a polynucleotide may encode a coding sequence of a protein, and/or comprise control or regulatory sequences of a coding sequence of a protein, such as enhancer or promoter sequences or terminator. A polynucleotide may for example comprise native coding sequence of a gene or their fragments, or variant sequences that have been optimized for optimal gene expression in a specific host cell (for example to take into account codon bias).
[0091] As used herein, the term, "optimized" with reference to a polynucleotide means that a polynucleotide has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, for example, a filamentous fungal cell such as a Trichoderma cell. Heterologous nucleotide sequences that are transfected in a host cell are typically optimized to retain completely or as much as possible the amino acid sequence originally encoded by the original (not optimized) nucleotide sequence. The optimized sequences herein have been engineered to have codons that are preferred in the corresponding production cell or organism, for example the filamentous fungal cell. The amino acid sequences encoded by optimized nucleotide sequences may also be referred to as optimized.
[0092] As used herein, a "peptide" or a "polypeptide" is an amino acid sequence including a plurality of consecutive polymerized amino acid residues. The peptide or polypeptide may include modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues. As used herein, a "protein" may refer to a peptide or a polypeptide or a combination of more than one peptide or polypeptide assembled together by covalent or non-covalent bonds. Unless specified, the term "protein" may encompass one or more amino acid sequences with their post-translation modifications, and in particular with either O-mannosylation or N-glycan modifications.
[0093] As used herein, the term "glycoprotein" refers to a protein which comprises at least one N-linked glycan attached to at least one asparagine residue of a protein, or at least one mannose attached to at least one serine or threonine resulting in O-mannosylation.
[0094] The terms "O-mannosylation" or "O-mannosyltransferase activity" are used herein to refer to the covalent linkage of at least one mannose to one specific amino acid via one oxygen (typically from serine or threonine). O-mannosyltransferase protein typically adds mannose to hydroxyl groups such as hydroxyl of serine or threonine residues.
[0095] In particular, O-mannosyltransferase activity may refer to the specificity of O-mannosyltransferase activity of fungal PMT gene encoding enzymes, and more specifically with the same specificity of T. reesei PMT1.
[0096] As used herein, "glycan" refers to an oligosaccharide chain that can be linked to a carrier such as an amino acid, peptide, polypeptide, lipid or a reducing end conjugate. In certain embodiments, the invention relates to N-linked glycans ("N-glycan") conjugated to a polypeptide N-glycosylation site such as -Asn-Xaa-Ser/Thr- by N-linkage to side-chain amide nitrogen of asparagine residue (Asn), where Xaa is any amino acid residue except Pro. The invention may further relate to glycans as part of dolichol-phospho-oligosaccharide (Dol-P-P-OS) precursor lipid structures, which are precursors of N-linked glycans in the endoplasmic reticulum of eukaryotic cells. The precursor oligosaccharides are linked from their reducing end to two phosphate residues on the dolichol lipid. For example, .alpha.3-mannosyltransferase Alg3 modifies the Dol-P-P-oligosaccharide precursor of N-glycans. Generally, the glycan structures described herein are terminal glycan structures, where the non-reducing residues are not modified by other monosaccharide residue or residues.
[0097] As used throughout the present disclosure, glycolipid and carbohydrate nomenclature is essentially according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 29). It is assumed that Gal (galactose), Glc (glucose), GlcNAc (N-acetylglucosamine), GalNAc (N-acetylgalactosamine), Man (mannose), and Neu5Ac are of the D-configuration, Fuc of the L-configuration, and all the monosaccharide units in the pyranose form (D-Galp, D-Glcp, D-GlcpNAc, D-GalpNAc, D-Manp, L-Fucp, D-Neup5Ac). The amine group is as defined for natural galactose and glucosamines on the 2-position of GalNAc or GlcNAc. Glycosidic linkages are shown partly in shorter and partly in longer nomenclature, the linkages of the sialic acid SA/Neu5X-residues .alpha.3 and .alpha.6 mean the same as .alpha.2-3 and .alpha.2-6, respectively, and for hexose monosaccharide residues .alpha.1-3, .alpha.1-6, .beta.1-2, .beta.1-3, .beta.1-4, and .beta.1-6 can be shortened as .alpha.3, .alpha.6, .beta.2, .beta.3, .beta.4, and .beta.6, respectively. Lactosamine refers to type II N-acetyllactosamine, Gal.beta.4GlcNAc, and/or type I N-acetyllactosamine. Gal.beta.3GlcNAc and sialic acid (SA) refer to N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), or any other natural sialic acid including derivatives of Neu5X. Sialic acid is referred to as NeuNX or Neu5X, where preferably X is Ac or Gc. Occasionally Neu5Ac/Gc/X may be referred to as NeuNAc/NeuNGc/NeuNX.
[0098] The sugars typically constituting N-glycans found in mammalian glycoprotein, include, without limitation, N-acetylglucosamine (abbreviated hereafter as "GlcNAc"), mannose (abbreviated hereafter as "Man"), glucose (abbreviated hereafter as "Glc"), galactose (abbreviated hereafter as "Gal"), and sialic acid (abbreviated hereafter as "Neu5Ac"). N-glycans share a common pentasaccharide referred to as the "core" structure Man.sub.3GlcNAc.sub.2 (Man.alpha.6(Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc, referred to as Man3). In some embodiments Man3 glycan or its derivative Man.alpha.6(GlcNAc.beta.2Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc is the major glycoform. When a fucose is attached to the core structure, preferably .alpha.6-linked to reducing end GlcNAc, the N-glycan or the core of N-glycan, may be represented as Man.sub.3GlcNAc.sub.2(Fuc). In an embodiment the major N-glycan is Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.4Glc- NAc (Man5).
[0099] Preferred hybrid type N-glycans comprise GlcNAc.beta.2Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4Gl- cNA.beta.4GlcNAc ("GlcNAcMan5"), or b4-galactosylated derivatives thereof Gal.beta.4GlcNAcMan3, G1, G2, or GalGlcNAcMan5 glycoform.
[0100] A "complex N-glycan" refers to a N-glycan which has at least one GlcNAc residue, optionally by GlcNAc.beta.2-residue, on terminal 1,3 mannose arm of the core structure and at least one GlcNAc residue, optionally by GlcNAc.beta.2-residue, on terminal 1,6 mannose arm of the core structure.
[0101] Such complex N-glycans include, without limitation, GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (also referred as G0 glycoform), Gal.sub.1GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (also referred as G1 glycoform), and Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (also referred as G2 glycoform), and their core fucosylated glycoforms FG0, FG1 and FG2, respectively GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc), Gal.sub.1GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc), and Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc).
[0102] "Increased" or "Reduced activity of an endogenous enzyme": The filamentous fungal cell may have increased or reduced levels of activity of various endogenous enzymes. A reduced level of activity may be provided by inhibiting the activity of the endogenous enzyme with an inhibitor, an antibody, or the like. In certain embodiments, the filamentous fungal cell is genetically modified in ways to increase or reduce activity of various endogenous enzymes. "Genetically modified" refers to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic host cell that expresses a polypeptide at elevated levels, at lowered levels, or in a mutated form. In other words, the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the cell to alter expression of a desired protein.
[0103] "Genetic modifications" which result in a decrease or deficiency in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) can be referred to as inactivation (complete or partial), knock-out, deletion, disruption, interruption, blockage, silencing, or down-regulation, or attenuation of expression of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such gene, can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete (disruption) or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity or action). More specifically, reference to decreasing the action of proteins discussed herein generally refers to any genetic modification in the host cell in question, which results in decreased expression and/or functionality (biological activity) of the proteins and includes decreased activity of the proteins (e.g., decreased catalysis), increased inhibition or degradation of the proteins as well as a reduction or elimination of expression of the proteins. For example, the action or activity of a protein can be decreased by blocking or reducing the production of the protein, reducing protein action, or inhibiting the action of the protein. Combinations of some of these modifications are also possible. Blocking or reducing the production of a protein can include placing the gene encoding the protein under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the protein (and therefore, of protein synthesis) could be turned off. Blocking or reducing the action of a protein could also include using an excision technology approach similar to that described in U.S. Pat. No. 4,743,546. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal.
[0104] In general, according to the present invention, an increase or a decrease in a given characteristic of a mutant or modified protein (e.g., enzyme activity) is made with reference to the same characteristic of a parent (i.e., normal, not modified) protein that is derived from the same organism (from the same source or parent sequence), which is measured or established under the same or equivalent conditions. Similarly, an increase or decrease in a characteristic of a genetically modified host cell (e.g., expression and/or biological activity of a protein, or production of a product) is made with reference to the same characteristic of a wild-type host cell of the same species, and preferably the same strain, under the same or equivalent conditions. Such conditions include the assay or culture conditions (e.g., medium components, temperature, pH, etc.) under which the activity of the protein (e.g., expression or biological activity) or other characteristic of the host cell is measured, as well as the type of assay used, the host cell that is evaluated, etc. As discussed above, equivalent conditions are conditions (e.g., culture conditions) which are similar, but not necessarily identical (e.g., some conservative changes in conditions can be tolerated), and which do not substantially change the effect on cell growth or enzyme expression or biological activity as compared to a comparison made under the same conditions.
[0105] Preferably, a genetically modified host cell that has a genetic modification that increases or decreases (reduces) the activity of a given protein (e.g., an O-mannosyltransferase or protease) has an increase or decrease, respectively, in the activity or action (e.g., expression, production and/or biological activity) of the protein, as compared to the activity of the protein in a parent host cell (which does not have such genetic modification), of at least about 5%, and more preferably at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 60%, 65%, 70%, 75 80%, 85 90%, 95%, or any percentage, in whole integers between 5% and 100% (e.g., 6%, 7%, 8%, etc.).
[0106] In another aspect of the invention, a genetically modified host cell that has a genetic modification that increases or decreases (reduces) the activity of a given protein (e.g., an O-mannosyltransferase or protease) has an increase or decrease, respectively, in the activity or action (e.g., expression, production and/or biological activity) of the protein, as compared to the activity of the wild-type protein in a parent host cell, of at least about 2-fold, and more preferably at least about 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or any whole integer increment starting from at least about 2-fold (e.g., 3-fold, 4-fold, 5-fold, 6-fold, etc.).
[0107] As used herein, the terms "identical" or percent "identity," in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.
[0108] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn, the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp, the default parameters are Gap opening penalty=11 and Gap extension penalty=1.
[0109] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions including, but not limited to from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85(8):2444-2448, 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.), or by manual alignment and visual inspection [see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].
[0110] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0111] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). 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, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[0112] "Functional variant" or "functional homologous gene" as used herein refers to a coding sequence or a protein having sequence similarity with a reference sequence, typically, at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% identity with the reference coding sequence or protein, and retaining substantially the same function as said reference coding sequence or protein. A functional variant may retain the same function but with reduced or increased activity. Functional variants include natural variants, for example, homologs from different species or artificial variants, resulting from the introduction of a mutation in the coding sequence. Functional variant may be a variant with only conservatively modified mutations.
[0113] "Conservatively modified mutations" as used herein include individual substitutions, deletions or additions to an encoded amino acid sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
Filamentous Fungal Cells
[0114] As used herein, "filamentous fungal cells" include cells from all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). Filamentous fungal cells are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
[0115] Preferably, the filamentous fungal cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., mammalian proteins), or the resulting intermediates. General methods to disrupt genes of and cultivate filamentous fungal cells are disclosed, for example, for Penicillium, in Kopke et al. (2010) Appl Environ Microbiol. 76(14):4664-74. doi: 10.1128/AEM.00670-10, for Aspergillus, in Maruyama and Kitamoto (2011), Methods in Molecular Biology, vol. 765, D0110.1007/978-1-61779-197-0_27; for Neurospora, in Collopy et al. (2010) Methods Mol Biol. 2010; 638:33-40. doi: 10.1007/978-1-60761-611-5_3; and for Myceliophthora or Chrysosporium PCT/NL2010/000045 and PCT/EP98/06496.
[0116] Examples of suitable filamentous fungal cells include, without limitation, cells from an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma/Hypocrea strain.
[0117] In certain embodiments, the filamentous fungal cell is from a Trichoderma sp., Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Chrysosporium, Chrysosporium lucknowense, Filibasidium, Fusarium, Gibberella, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, or Tolypocladium strain.
[0118] In some embodiments, the filamentous fungal cell is a Myceliophthora or Chrysosporium, Neurospora or Trichoderma strain.
[0119] Aspergillus fungal cells of the present disclosure may include, without limitation, Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus terreus.
[0120] Neurospora fungal cells of the present disclosure may include, without limitation, Neurospora crassa.
[0121] Myceliophthora fungal cells of the present disclosure may include, without limitation, Myceliophthora thermophila.
[0122] In a preferred embodiment, the filamentous fungal cell is a Trichoderma fungal cell. Trichoderma fungal cells of the present disclosure may be derived from a wild-type Trichoderma strain or a mutant thereof. Examples of suitable Trichoderma fungal cells include, without limitation, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichoderma virens, Trichoderma viride; and alternative sexual form thereof (i.e., Hypocrea).
[0123] In a more preferred embodiment, the filamentous fungal cell is a Trichoderma reesei, and for example, strains derived from ATCC 13631 (QM 6a), ATCC 24449 (radiation mutant 207 of QM 6a), ATCC 26921 (QM 9414; mutant of ATCC 24449), VTT-D-00775 (Selinheimo et al., FEBS J., 2006, 273: 4322-4335), Rut-C30 (ATCC 56765), RL-P37 (NRRL 15709) or T. harzianum isolate T3 (Wolffhechel, H., 1989).
[0124] The invention described herein relates to a PMT deficient filamentous fungal cell, for example selected from Trichoderma, Neurospora, Myceliophthora or a Chrysosporium cells, such as Trichoderma reesei fungal cell, comprising:
[0125] a. at least a first mutation that reduces or eliminates an endogenous protease activity compared to the parental filamentous fungal cell which does not have said first mutation (i.e. a protease-deficient mutation), and,
[0126] b. at least a second mutation in a PMT gene that reduces or eliminates an endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation (i.e. a PMT-deficient mutation). Proteases with Reduced Activity
[0127] It has been found that reducing protease activity enables to increase substantially the production of heterologous mammalian protein. Indeed, such proteases found in filamentous fungal cells that express a heterologous protein normally catalyse significant degradation of the expressed recombinant protein. Thus, by reducing the activity of proteases in filamentous fungal cells that express a heterologous protein, the stability of the expressed protein is increased, resulting in an increased level of production of the protein, and in some circumstances, improved quality of the produced protein (e.g., full-length instead of degraded).
[0128] Proteases include, without limitation, aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases, and sedolisin proteases. Such proteases may be identified and isolated from filamentous fungal cells and tested to determine whether reduction in their activity affects the production of a recombinant polypeptide from the filamentous fungal cell. Methods for identifying and isolating proteases are well known in the art, and include, without limitation, affinity chromatography, zymogram assays, and gel electrophoresis. An identified protease may then be tested by deleting the gene encoding the identified protease from a filamentous fungal cell that expresses a recombinant polypeptide, such a heterologous or mammalian polypeptide, and determining whether the deletion results in a decrease in total protease activity of the cell, and an increase in the level of production of the expressed recombinant polypeptide. Methods for deleting genes, measuring total protease activity, and measuring levels of produced protein are well known in the art and include the methods described herein.
Aspartic Proteases
[0129] Aspartic proteases are enzymes that use an aspartate residue for hydrolysis of the peptide bonds in polypeptides and proteins. Typically, aspartic proteases contain two highly-conserved aspartate residues in their active site which are optimally active at acidic pH. Aspartic proteases from eukaryotic organisms such as Trichoderma fungi include pepsins, cathepsins, and renins. Such aspartic proteases have a two-domain structure, which is thought to arise from ancestral gene duplication. Consistent with such a duplication event, the overall fold of each domain is similar, though the sequences of the two domains have begun to diverge. Each domain contributes one of the catalytic aspartate residues. The active site is in a cleft formed by the two domains of the aspartic proteases. Eukaryotic aspartic proteases further include conserved disulfide bridges, which can assist in identification of the polypeptides as being aspartic acid proteases.
[0130] Nine aspartic proteases have been identified in Trichoderma fungal cells: pep1 (tre74156); pep2 (tre53961); pep3 (tre121133); pep4 (tre77579), pep5 (tre81004), and pep7 (tre58669), pep8 (tre122076), pep11 (121306), and pep12 (tre119876).
[0131] Examples of suitable aspartic proteases include, without limitation, Trichoderma reesei pep1 (SEQ ID NO: 22), Trichoderma reesei pep2 (SEQ ID NO: 18), Trichoderma reesei pep3 (SEQ ID NO: 19); Trichoderma reesei pep4 (SEQ ID NO: 20), Trichoderma reesei pep5 (SEQ ID NO: 21) and Trichoderma reesei pep7 (SEQ ID NO:23), Trichoderma reesei EGR48424 pep8 (SEQ ID NO:134), Trichoderma reesei EGR49498 pep11 (SEQ ID NO:135), Trichoderma reesei EGR52517 pep12 (SEQ ID NO:35), and homologs thereof. Examples of homologs of pep1, pep2, pep3, pep4, pep5, pep7, pep8, pep11 and pep12 proteases identified in other organisms are also described in PCT/EP/2013/050186, the content of which being incorporated by reference.
Trypsin-Like Serine Proteases
[0132] Trypsin-like serine proteases are enzymes with substrate specificity similar to that of trypsin. Trypsin-like serine proteases use a serine residue for hydrolysis of the peptide bonds in polypeptides and proteins. Typically, trypsin-like serine proteases cleave peptide bonds following a positively-charged amino acid residue. Trypsin-like serine proteases from eukaryotic organisms such as Trichoderma fungi include trypsin 1, trypsin 2, and mesotrypsin. Such trypsin-like serine proteases generally contain a catalytic triad of three amino acid residues (such as histidine, aspartate, and serine) that form a charge relay that serves to make the active site serine nucleophilic. Eukaryotic trypsin-like serine proteases further include an "oxyanion hole" formed by the backbone amide hydrogen atoms of glycine and serine, which can assist in identification of the polypeptides as being trypsin-like serine proteases.
[0133] One trypsin-like serine protease has been identified in Trichoderma fungal cells: tsp1 (tre73897). As discussed in PCT/EP/2013/050186, tsp1 has been demonstrated to have a significant impact on expression of recombinant glycoproteins, such as immunoglobulins.
[0134] Examples of suitable tsp1 proteases include, without limitation, Trichoderma reesei tsp1 (SEQ ID NO: 24) and homologs thereof. Examples of homologs of tsp1 proteases identified in other organisms are described in PCT/EP/2013/050186.
Subtilisin Proteases
[0135] Subtilisin proteases are enzymes with substrate specificity similar to that of subtilisin. Subtilisin proteases use a serine residue for hydrolysis of the peptide bonds in polypeptides and proteins. Generally, subtilisin proteases are serine proteases that contain a catalytic triad of the three amino acids aspartate, histidine, and serine. The arrangement of these catalytic residues is shared with the prototypical subtilisin from Bacillus licheniformis. Subtilisin proteases from eukaryotic organisms such as Trichoderma fungi include furin, MBTPS1, and TPP2. Eukaryotic trypsin-like serine proteases further include an aspartic acid residue in the oxyanion hole.
[0136] Seven subtilisin proteases have been identified in Trichoderma fungal cells: slp1 (tre51365); slp2 (tre123244); slp3 (tre123234); slp5 (tre64719), slp6 (tre121495), slp7 (tre123865), and slp8 (tre58698). Subtilisin protease slp7 resembles also sedolisin protease tpp1.
[0137] Examples of suitable slp proteases include, without limitation, Trichoderma reesei slp1 (SEQ ID NO: 25), slp2 (SEQ ID NO: 26); slp3 (SEQ ID NO: 27); slp5 (SEQ ID NO: 28), slp6 (SEQ ID NO: 29), slp7 (SEQ ID NO: 30), and slp8 (SEQ ID NO: 31), and homologs thereof. Examples of homologs of slp proteases identified in other organisms are described in PCT/EP/2013/050186.
Glutamic Proteases
[0138] Glutamic proteases are enzymes that hydrolyse the peptide bonds in polypeptides and proteins. Glutamic proteases are insensitive to pepstatin A, and so are sometimes referred to as pepstatin insensitive acid proteases. While glutamic proteases were previously grouped with the aspartic proteases and often jointly referred to as acid proteases, it has been recently found that glutamic proteases have very different active site residues than aspartic proteases.
[0139] Two glutamic proteases have been identified in Trichoderma fungal cells: gap1 (tre69555) and gap2 (tre106661). Examples of suitable gap proteases include, without limitation, Trichoderma reesei gap1 (SEQ ID NO: 32), Trichoderma reesei gap2 (SEQ ID NO: 33), and homologs thereof. Examples of homologs of gap proteases identified in other organisms are described in PCT/EP/2013/050186.
Sedolisin Proteases and Homologs of Proteases
[0140] Sedolisin proteases are enzymes that use a serine residue for hydrolysis of the peptide bonds in polypeptides and proteins. Sedolisin proteases generally contain a unique catalytic triad of serine, glutamate, and aspartate. Sedolisin proteases also contain an aspartate residue in the oxyanion hole. Sedolisin proteases from eukaryotic organisms such as Trichoderma fungi include tripeptidyl peptidase.
[0141] Examples of suitable tpp1 proteases include, without limitation, Trichoderma reesei tpp1 tre82623 (SEQ ID NO: 34) and homologs thereof. Examples of homologs of tpp1 proteases identified in other organisms are described in PCT/EP/2013/050186.
[0142] As used in reference to protease, the term "homolog" refers to a protein which has protease activity and exhibit sequence similarity with a known (reference) protease sequence. Homologs may be identified by any method known in the art, preferably, by using the BLAST tool to compare a reference sequence to a single second sequence or fragment of a sequence or to a database of sequences. As described in the "Definitions" section, BLAST will compare sequences based upon percent identity and similarity.
[0143] Preferably, a homologous protease has at least 30% identity with (optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared to one of the protease sequences listed above, including T. reesei pep1, pep2, pep3, pep4, pep5, pep7, pep8, pep 11, pep 12, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2. Corresponding homologous proteases from N. crassa and M. thermophila are shown in SEQ ID NO: 136-169.
Reducing the Activity of Proteases in the Filamentous Fungal Cell of the Invention
[0144] The filamentous fungal cells according to the invention have reduced activity of at least one endogenous protease, typically 2, 3, 4, 5 or more, in order to improve the stability and production of the protein with reduced O-mannosylation in said filamentous fungal cell, preferably in a PMT-deficient Trichoderma cell.
[0145] The activity of proteases found in filamentous fungal cells can be reduced by any method known to those of skill in the art. In some embodiments reduced activity of proteases is achieved by reducing the expression of the protease, for example, by promoter modification or RNAi.
[0146] In further embodiments, the reduced or eliminated expression of the proteases is the result of anti-sense polynucleotides or RNAi constructs that are specific for each of the genes encoding each of the proteases. In one embodiment, an RNAi construct is specific for a gene encoding an aspartic protease such as a pep-type protease, a trypsin-like serine proteases such as a tsp1, a glutamic protease such as a gap-type protease, a subtilisin protease such as a slp-type protease, or a sedolisin protease such as a tpp1 or a slp7 protease. In one embodiment, an RNAi construct is specific for the gene encoding a slp-type protease. In one embodiment, an RNAi construct is specific for the gene encoding slp2, slp3, slp5 or slp6. In one embodiment, an RNAi construct is specific for two or more proteases. In one embodiment, two or more proteases are any one of the pep-type proteases, any one of the trypsin-like serine proteases, any one of the slp-type proteases, any one of the gap-type proteases and/or any one of the sedolisin proteases. In one embodiment, two or more proteases are slp2, slp3, slp5 and/or slp6. In one embodiment, RNAi construct comprises any one of the following nucleic acid sequences (see also PCT/EP/2013/050186).
TABLE-US-00001 RNAi Target sequence GCACACTTTCAAGATTGGC (SEQ ID NO: 15) GTACGGTGTTGCCAAGAAG (SEQ ID NO: 16) GTTGAGTACATCGAGCGCGACAGCATTGTGCACACCATGCTTCCCCTCGA GTCCAAGGACAGCATCATCGTTGAGGACTCGTGCAACGGCGAGACGGAGA AGCAGGCTCCCTGGGGTCTTGCCCGTATCTCTCACCGAGAGACGCTCAAC TTTGGCTCCTTCAACAAGTACCTCTACACCGCTGATGGTGGTGAGGGTGT TGATGCCTATGTCATTGACACCGGCACCAACATCGAGCACGTCGACTTTG AGGGTCGTGCCAAGTGGGGCAAGACCATCCCTGCCGGCGATGAGGACGAG GACGGCAACGGCCACGGCACTCACTGCTCTGGTACCGTTGCTGGTAAGAA GTACGGTGTTGCCAAGAAGGCCCACGTCTACGCCGTCAAGGTGCTCCGAT CCAACGGATCCGGCACCATGTCTGACGTCGTCAAGGGCGTCGAGTACG (SEQ ID NO: 17)
[0147] In other embodiments, reduced activity of proteases is achieved by modifying the gene encoding the protease. Examples of such modifications include, without limitation, a mutation, such as a deletion or disruption of the gene encoding said endogenous protease activity.
[0148] Accordingly, the invention relates to a filamentous fungal cell, such as a PMT-deficient Trichoderma cell, which has a first mutation that reduces or eliminates at least one endogenous protease activity compared to a parental filamentous fungal cell which does not have such protease deficient mutation, said filamentous fungal cell further comprising at least a second mutation in a PMT gene that reduces endogenous protein O-mannosyltransferase activity compared to a parental Trichoderma cell which does not have said second PMT-deficient mutation.
[0149] Deletion or disruption mutation includes without limitation knock-out mutation, a truncation mutation, a point mutation, a missense mutation, a substitution mutation, a frameshift mutation, an insertion mutation, a duplication mutation, an amplification mutation, a translocation mutation, or an inversion mutation, and that results in a reduction in the corresponding protease activity. Methods of generating at least one mutation in a protease encoding gene of interest are well known in the art and include, without limitation, random mutagenesis and screening, site-directed mutagenesis, PCR mutagenesis, insertional mutagenesis, chemical mutagenesis, and irradiation.
[0150] In certain embodiments, a portion of the protease encoding gene is modified, such as the region encoding the catalytic domain, the coding region, or a control sequence required for expression of the coding region. Such a control sequence of the gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, without limitation, a leader sequence, a propeptide sequence, a signal sequence, a transcription terminator, and a transcriptional activator.
[0151] Protease encoding genes that are present in filamentous fungal cells may also be modified by utilizing gene deletion techniques to eliminate or reduce expression of the gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.
[0152] The protease encoding genes that are present in filamentous fungal cells may also be modified by introducing, substituting, and/or removing one or more nucleotides in the gene, or a control sequence thereof required for the transcription or translation of the gene. For example, nucleotides may be inserted or removed for the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by methods known in the art, including without limitation, site-directed mutagenesis and peR generated mutagenesis (see, for example, Botstein and Shortie, 1985, Science 229: 4719; Lo et al., 1985, Proceedings of the National Academy of Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids Research 16: 7351; Shimada, 1996, Meth. Mol. Bioi. 57: 157; Ho et al., 1989, Gene 77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar and Sommer, 1990, BioTechniques 8: 404).
[0153] Additionally, protease encoding genes that are present in filamentous fungal cells may be modified by gene disruption techniques by inserting into the gene a disruptive nucleic acid construct containing a nucleic acid fragment homologous to the gene that will create a duplication of the region of homology and incorporate construct nucleic acid between the duplicated regions. Such a gene disruption can eliminate gene expression if the inserted construct separates the promoter of the gene from the coding region or interrupts the coding sequence such that a nonfunctional gene product results. A disrupting construct may be simply a selectable marker gene accompanied by 5' and 3' regions homologous to the gene. The selectable marker enables identification of transformants containing the disrupted gene.
[0154] Protease encoding genes that are present in filamentous fungal cells may also be modified by the process of gene conversion (see, for example, Iglesias and Trautner, 1983, Molecular General Genetics 189:5 73-76). For example, in the gene conversion a nucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective nucleotide sequence, which is then transformed into a Trichoderma strain to produce a defective gene. By homologous recombination, the defective nucleotide sequence replaces the endogenous gene. It may be desirable that the defective nucleotide sequence also contains a marker for selection of transformants containing the defective gene.
[0155] Protease encoding genes of the present disclosure that are present in filamentous fungal cells that express a recombinant polypeptide may also be modified by established anti-sense techniques using a nucleotide sequence complementary to the nucleotide sequence of the gene (see, for example, Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). In particular, expression of the gene by filamentous fungal cells may be reduced or inactivated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which may be transcribed in the strain and is capable of hybridizing to the mRNA produced in the cells. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.
[0156] Protease encoding genes that are present in filamentous fungal cells may also be modified by random or specific mutagenesis using methods well known in the art, including without limitation, chemical mutagenesis (see, for example, Hopwood, The Isolation of Mutants in Methods in Microbiology (J. R. Norris and D. W. Ribbons, eds.) pp. 363-433, Academic Press, New York, 25 1970). Modification of the gene may be performed by subjecting filamentous fungal cells to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or inactivated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, subjecting the DNA sequence to peR generated mutagenesis, or any combination thereof. Examples of physical and chemical mutagenizing agents include, without limitation, ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the filamentous fungal cells, such as Trichoderma cells, to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and then selecting for mutants exhibiting reduced or no expression of the gene.
[0157] In certain embodiments, the at least one mutation or modification in a protease encoding gene of the present disclosure results in a modified protease that has no detectable protease activity. In other embodiments, the at least one modification in a protease encoding gene of the present disclosure results in a modified protease that has at least 25% less, at least 50% less, at least 75% less, at least 90%, at least 95%, or a higher percentage less protease activity compared to a corresponding non-modified protease.
[0158] The filamentous fungal cells or Trichoderma fungal cells of the present disclosure may have reduced or no detectable protease activity of at least three, or at least four proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, gap1 and gap2. In preferred embodiment, a filamentous fungal cell according to the invention is a PMT-deficient filamentous fungal cell which has a deletion or disruption in at least 3 or 4 endogenous proteases, resulting in no detectable activity for such deleted or disrupted endogenous proteases and further comprising another mutation in a PMT gene that reduces endogenous protein O-mannosyltransferase activity compared to a parental Trichoderma cell which does not have said mutation.
[0159] In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in pep1, tsp1, and slp1. In other embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in gap1, slp1, and pep1. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1 and gap1. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1 and pep4. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4 and slp1. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, and slp3. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, and pep3. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3 and pep2. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2 and pep5. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5 and tsp1. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1 and slp7. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1, slp7 and slp8. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1, slp7, slp8 and gap2. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in at least three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp2, slp3, slp7, gap1 and gap2. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in at least three to six endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2. In certain embodiments, the PMT-deficient filamentous fungal cell or Trichoderma cell, has reduced or no detectable protease activity in at least seven to ten endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.
[0160] In one embodiment that may be combined with the precedent embodiments, the filamentous fungal cell of the invention does not comprise a deletion or disruption of an endogenous gene encoding a chaperone protein. In particular, said filamentous fungal cell of the invention expresses functional endogenous chaperone protein Protein Disulphide Isomerase (PDI).
Endogenous O-Mannosyltransferase in Filamentous Fungal Cells
[0161] O-mannosyltransferases are encoded by pmt genes in yeasts and filamentous fungi, which can be divided into three subfamilies, based on sequence homologies: PMT1, PMT2 and PMT4.
[0162] For example, in yeast S. cerevisiae, 7 different PMTs have been characterized: ScPMT1, ScPMT5 and ScPMT7 belong to the PMT1 subfamily. ScPMT2, ScPMT3 and ScPMT6 belong to the PMT2 subfamily and ScPMT4 belongs to the PMT4 subfamily. Such O-mannosyltransferases and their coding sequences may be identified and isolated from filamentous fungal cells and tested to determine whether reduction in their activity enables the reduction of O-mannosylation on secreted O-mannosylated recombinant protein preferably not affecting the production of such recombinant polypeptide from the filamentous fungal cell. Methods for identifying and isolating PMTs are well known in the art. An identified O-mannosyltransferase may then be tested by deleting the gene encoding the identified O-mannosyltransferase from a filamentous fungal cell that expresses a recombinant O-mannosylated protein, such a heterologous or mammalian O-mannosylated protein, and determining whether the deletion results in a decrease in total O-mannosyltransferase activity of the cell, preferably not affecting the level of production of the expressed recombinant protein. Methods for deleting genes and measuring levels of produced protein are well known in the art and include the methods described herein.
[0163] Three O-mannosyltransferases have been identified in Trichoderma fungal cells: pmt1, pmt2 and pmt3, belonging respectively based on sequence homologies to the PMT4, PMT1 and PMT2 subfamily.
[0164] Examples of suitable O-mannosyltransferase include, without limitation, Trichoderma reesei pmt1 (SEQ ID NO: 2), Trichoderma reesei pmt2 (SEQ ID NO: 3), Trichoderma reesei pmt3 (SEQ ID NO: 4) and homologs thereof. FIG. 5 shows phylogeny of pmt homologs in selected filamentous fungi and FIG. 6 shows an alignment of pmt1 conserved domains among different species.
[0165] In a preferred embodiment, said PMT-deficient filamentous fungal cell, e.g., a Trichoderma cell, has at least one mutation in a PMT gene selected from the group consisting of:
[0166] a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,
[0167] b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, and,
[0168] c) a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.
[0169] More preferably, said PMT-deficient filamentous fungal cell, e.g., a Trichoderma cell, has at least one mutation in a PMT gene which
[0170] a) has a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, and,
[0171] b) is capable of restoring, at least 50%, preferably about 100% of parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in a T. reesei PMT1 gene.
[0172] Methods for disrupting PMT1 gene in T. reesei are disclosed in the Examples below.
[0173] Sequences of homologs of pmt1 in filamentous fungi can be found in the databases using sequence alignment search tools, such as BLAST algorithm. It includes without limitation, A. oryzae gi391865791, EIT75070.1 (SEQ ID NO:5), A. niger gi317036343, XP_001398147.2 (SEQ ID NO:6), A. nidulans gi67522004, XP_659063.1 (SEQ ID NO:7), T. virens gi358379774, EHK17453.1 (SEQ ID NO:8), T. atroviride gi358400594, EHK49920.1 (SEQ ID NO:9), F. oxysporum gi342879728, EGU80965.1 (SEQ ID NO:10), G. zeae gi46107450, XP_380784.1 (SEQ ID NO:11), M. thermophila gi367020262, XP_003659416.1 (SEQ ID NO:12), N. crassa gi164423013, XP_963926.2 (SEQ ID NO:13), and P. chrysogenum gi255953619, XP_002567562.1 (SEQ ID NO:14).
Reducing Endogenous Protein O-Mannosyltransferase Activity in Filamentous Fungal Cell of the Invention
[0174] The PMT-deficient filamentous fungal cells according to the invention have reduced activity of at least one O-mannosyltransferase activity, in order to reduce or decrease O-mannosylation in said filamentous fungal cell, preferably Trichoderma cell.
[0175] The activity of said O-mannosyltransferases found in filamentous fungal cells can be reduced by any method known to those of skill in the art. In some embodiments reduced activity of O-mannosyltransferases is achieved by reducing the expression of the O-mannosyltransferases, for example, by promoter modification or RNAi.
[0176] In other embodiments, reduced activity of O-mannosyltransferases is achieved by modifying the gene encoding the O-mannosyltransferase. Examples of such modifications include, without limitation, a mutation, such as a deletion or disruption of the gene encoding said endogenous O-mannosyltransferase activity.
[0177] Deletion or disruption mutation can be performed as described in the above sections, in particular in relation to deletion or disruption of genes encoding proteases. These includes without limitation knock-out mutation, a truncation mutation, a point mutation, a missense mutation, a substitution mutation, a frameshift mutation, an insertion mutation, a duplication mutation, an amplification mutation, a translocation mutation, or an inversion mutation, and that results in a reduction in the corresponding O-mannosyltransferase activity.
[0178] In certain embodiments, the mutation or modification in an O-mannosyltransferase (PMT) encoding gene of the present disclosure results in a modified O-mannosyltransferase that has no detectable O-mannosyltransferase activity. In other embodiments, the at least one modification in a O-mannosyltransferase encoding gene of the present disclosure results in a modified O-mannosyltransferase that has at least 25% less, at least 50% less, at least 75% less, at least 90%, at least 95%, or a higher percentage less O-mannosyltransferase activity compared to a corresponding non-modified O-mannosyltransferase.
[0179] In preferred embodiment, a mutation that reduces endogenous protein O-mannosyltransferase activity in a PMT-deficient filamentous fungal cell, e.g. Trichoderma cell, is a PMT-deficient cell which has a deletion or disruption of a PMT gene encoding said O-mannosyltransferase activity, resulting in no detectable expression for such deleted or disrupted PMT gene.
[0180] One specific embodiment of the present invention is a PMT-deficient Trichoderma reesei cell, comprising
[0181] a. at least a first mutation that reduces an endogenous protease activity compared to a parental Trichoderma cell which does not have said first mutation, and,
[0182] b. at least a disruption or deletion of PMT1 gene of T. reesei.
[0183] c. optionally, said cell further express a heterologous protein with serine or threonine, which has reduced O-mannosylation due to said mutation in said PMT gene.
[0184] The reduction (or decrease) of O-mannosyltransferase activity may be determined by comparing the O-mannosylation level of a heterologous protein in PMT-deficient filamentous fungal cell according to the invention, with the O-mannosylation level of a heterologous protein in the parental cell which does not have said PMT-deficient mutation.
[0185] In specific embodiments, the PMT-deficient filamentous fungal cell according to the invention expresses a heterologous protein which has reduced O-mannosylation due to said mutation in said PMT gene and the O-mannosylation level on the expressed heterologous protein is at least 20%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the O-mannosylation level of the heterologous protein when expressed in the parental filamentous fungal cell which does not have said second PMT-deficient mutation.
[0186] O-mannosylation level may also be determined as mole % of O-mannosylated polypeptide per total polypeptide as produced by the host cell of the invention. Analytical methods, such as MALDI TOF MS analysis may be used to determine O-mannosylation level as described in detail in the Example 1 below, section entitled "Analyses of Dpmt1 strains M403, M404, M406 and M407. In brief, a polypeptide as produced by the PMT-deficient filamentous fungal cell is purified to determine its O-mannoslyation level. Non O-mannosylated, and O-mannosylated structure of the polypeptide are separated and quantified by MALDI-TOF MS analysis. For example, the quantification of O-mannosylation level may be performed by determining area values or intensity of the different peaks of MALDI-TOF MS spectrum. An O-mannosylation level of 5% as determined by such method, using area values or intensity, reflects that about 95% (mol %) of the analysed polypeptides in the composition are not O-mannosylated In specific embodiments, the PMT-deficient filamentous fungal cell expresses a heterologous protein which has reduced O-mannosylation due to said mutation in said PMT gene, and the O-mannosylation level on the expressed heterologous protein (for example, as defined above by determining area or intensity values of MALDI TOF MS spectrum peaks) is reduced to less than 25%, 20%, 17%, 15%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or 0.5% (as mole % of mannose residues per polypeptide chain).
[0187] In an embodiment, the heterologous protein with reduced O-mannosylation is selected from the group consisting of:
[0188] a) an immunoglubulin, such as IgG,
[0189] b) a light chain or heavy chain of an immunoglobulin,
[0190] c) a heavy chain or a light chain of an antibody,
[0191] d) a single chain antibody,
[0192] e) a camelid antibody,
[0193] f) a monomeric or multimeric single domain antibody,
[0194] g) a FAb-fragment, a FAb2-fragment, and,
[0195] h) their antigen-binding fragments.
[0196] In a specific embodiment, a mutation that reduces endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding said engogenous protein O-mannosyltransferase activity. For example in Trichoderma cell, a mutation that reduces endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT1 gene.
Filamentous Fungal Cell for Producing Glycoproteins with Reduced O-Mannosylation and Mammalian-Like N-Glycans
[0197] The filamentous fungal cells according to the present invention may be useful in particular for producing heterologous glycoproteins with reduced O-mannosylation and mammalian-like N-glycans, such as complex N-glycans.
[0198] Accordingly, in one aspect, the filamentous fungal cell is further genetically modified to produce a mammalian-like N-glycan, thereby enabling in vivo production of glycoprotein with no or reduced O-mannosylation and with mammalian-like N-glycan as major glycoforms.
[0199] In certain embodiments, this aspect includes methods of producing glycoproteins with mammalian-like N-glycans in a Trichoderma cell.
[0200] In certain embodiment, the glycoprotein comprises, as a major glycoform, the mammalian-like N-glycan having the formula [(Gal.beta.4).sub.xGlcNAc.beta.2].sub.zMan.alpha.3([(Gal.beta.4).sub.yGlc- NAc.beta.2].sub.wMan.alpha.6)Man(.beta.4GlcNAc.beta.GlcNAc, where ( ) defines a branch in the structure, where [ ] or { } define a part of the glycan structure either present or absent in a linear sequence, and where x, y, z and w are 0 or 1, independently. In an embodiment w and z are 1.
[0201] In certain embodiments, the glycoprotein comprises, as a major glycoform, mammalian-like N-glycan selected from the group consisting of:
[0202] i. Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.4Glc- NAc (Man5 glycoform);
[0203] ii. GlcNAc.beta.2Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4Gl- cNA.beta.4GlcNAc (GlcNAcMan5 glycoform);
[0204] iii. Man.alpha.6(Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc (Man3 glycoform);
[0205] iv. Man.alpha.6(GlcNAc.beta.2Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc (GlcNAcMan3) or,
[0206] v. complex type N-glycans selected from the G0, G1, or G2 glycoform.
[0207] In an embodiment, the glycoprotein composition with mammalian-like N-glycans, preferably produced by an alg3 knock-out strain, include glycoforms that essentially lack or are devoid of glycans Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.4Glc- NAc (Man5). In specific embodiments, the filamentous fungal cell produces glycoproteins with, as major glycoform, the trimannosyl N-glycan structure Man.alpha.3[Man.alpha.6]Man.beta.4GlcNAc.beta.4GlcNAc. In other embodiments, the filamentous fungal cell procudes glycoproteins with, as major glycoform, the G0 N-glycan structure GlcNAc.beta.2Man.alpha.3[GlcNAc.beta.2Man.alpha.6]Man.beta.4GlcNAc.beta.4- GlcNAc.
[0208] In certain embodiments, the PMT-deficient filamentous fungal cell of the invention produces glycoprotein composition with a mixture of different N-glycans.
[0209] In some embodiments, Man3GlcNAc2 N-glycan (i.e. Man.alpha.3[Man.alpha.6]Man.beta.4GlcNAc.beta.4GlcNAc) represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0210] In other embodiments, GlcNAc2Man3 N-glycan (for example G0 GlcNAc.beta.2Man.alpha.3[GlcNAc.beta.2Man.alpha.6]Man.beta.4GlcNAc.beta.4- GlcNAc) represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0211] In other embodiments, GalGlcNAc2Man3GlcNAc2 N-glycan (for example G1 N-glycan) represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0212] In other embodiments, GaI2GlcNAc2Man3GlcNAc2 N-glycan (for example G2 N-glycan) represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0213] In other embodiments, complex type N-glycan represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0214] In other embodiments, hybrid type N-glycan represents at least 10%, at least 20%, at least at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of total (mol %) neutral N-glycans of a heterologous protein with reduced O-mannosylation, as expressed in a filamentous fungal cells of the invention.
[0215] In other embodiments, less than 0.5%, 0.1%, 0.05%, or less than 0.01% of the N-glycan of the glycoprotein composition produced by the host cell of the invention, comprises galactose. In certain embodiments, none of N-glycans comprise galactose.
[0216] The Neu5Gc and Gal.alpha.- (non-reducing end terminal Gal.alpha.3Gal.beta.4GlcNAc) structures are known xenoantigenic (animal derived) modifications of antibodies which are produced in animal cells such as CHO cells. The structures may be antigenic and, thus, harmful even at low concentrations. The filamentous fungi of the present invention lack biosynthetic pathways to produce the terminal Neu5Gc and Gal.alpha.- structures. In an embodiment that may be combined with the preceding embodiments less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/or O-glycans of the glycoprotein composition comprises Neu5Gc and/or Gal.alpha.- structure. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/or O-glycans of the antibody composition comprises Neu5Gc and/or Gal.alpha.- structure.
[0217] The filamentous fungal cells of the present invention lack genes to produce fucosylated heterologous proteins. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the glycoprotein composition comprises core fucose structures. In an embodiment that may be combined with the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the antibody composition comprises core fucose structures.
[0218] The terminal Gal.beta.4GlcNAc structure of N-glycan of mammalian cell produced glycans affects bioactivity of antibodies and Gal.beta.3GlcNAc may be xenoantigen structure from plant cell produced proteins. In an embodiment that may be combined with one or more of the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of N-glycan of the glycoprotein composition comprises terminal galactose epitopes Gal.beta.3/4GlcNAc. In an embodiment that may be combined with one or more of the preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the antibody composition comprises terminal galactose epitopes Gal.beta.3/4GlcNAc.
[0219] Glycation is a common post-translational modification of proteins, resulting from the chemical reaction between reducing sugars such as glucose and the primary amino groups on protein. Glycation occurs typically in neutral or slightly alkaline pH in cell cultures conditions, for example, when producing antibodies in CHO cells and analysing them (see, for example, Zhang et al. (2008) Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody. Anal Chem. 80(7):2379-2390). As filamentous fungi of the present invention are typically cultured in acidic pH, occurrence of glycation is reduced. In an embodiment that may be combined with the preceding embodiments, less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the glycoprotein composition comprises glycation structures. In an embodiment that may be combined with the preceding embodiments, less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the antibody composition comprises glycation structures.
[0220] In one embodiment, the glycoprotein composition, such as an antibody is devoid of one, two, three, four, five, or six of the structures selected from the group of Neu5Gc, terminal Gal.alpha.3Gal.beta.4GlcNAc, terminal Gal.beta.4GlcNAc, terminal Gal.beta.3GlcNAc, core linked fucose and glycation structures.
[0221] In certain embodiments, such glycoprotein protein with mammalian-like N-glycan and reduced O-mannosylation, as produced in the filamentous fungal cell of the invention, is a therapeutic protein. Therapeutic proteins may include immunoglobulin, or a protein fusion comprising a Fc fragment or other therapeutic glycoproteins, such as antibodies, erythropoietins, interferons, growth hormones, albumins or serum albumin, enzymes, or blood-clotting factors and may be useful in the treatment of humans or animals. For example, the glycoproteins with mammalian-like N-glycan and reduced O-mannosylation as produced by the filamentous fungal cell according to the invention may be a therapeutic glycoprotein such as rituximab.
[0222] Methods for producing glycoproteins with mammalian-like N-glycans in filamentous fungal cells are also described for example in WO2012/069593.
[0223] In one aspect, the filamentous fungal cell according to the invention as described above, is further genetically modified to mimic the traditional pathway of mammalian cells, starting from Man5 N-glycans as acceptor substrate for GnTI, and followed sequentially by GnT1, mannosidase II and GnTII reaction steps (hereafter referred as the "traditional pathway" for producing G0 glycoforms). In one variant, a single recombinant enzyme comprising the catalytic domains of GnTI and GnTII, is used.
[0224] Alternatively, in a second aspect, the filamentous fungal cell according to the invention as described above is further genetically modified to have alg3 reduced expression, allowing the production of core Man.sub.3GlcNAc.sub.2 N-glycans, as acceptor substrate for GnTI and GnTII subsequent reactions and bypassing the need for mannosidase .alpha.1,2 or mannosidase II enzymes (the reduced "alg3" pathway). In one variant, a single recombinant enzyme comprising the catalytic domains of GnTI and GnTII, is used.
[0225] In such embodiments for mimicking the traditional pathway for producing glycoproteins with mammalian-like N-glycans, a Man.sub.5 expressing filamentous fungal cell, such as T. reesei strain, may be transformed with a GnTI or a GnTII/GnTI fusion enzyme using random integration or by targeted integration to a known site known not to affect Man5 glycosylation. Strains that synthesise GlcNAcMan5 N-glycan for production of proteins having hybrid type glycan(s) are selected. The selected strains are further transformed with a catalytic domain of a mannosidase II-type mannosidase capable of cleaving Man5 structures to generate GlcNAcMan3 for production of proteins having the corresponding GlcNAcMan3 glycoform or their derivative(s). In certain embodiments, mannosidase II-type enzymes belong to glycoside hydrolase family 38 (cazy.org/GH38_all.html). Characterized enzymes include enzymes listed in cazy.org/GH38_characterized.html. Especially useful enzymes are Golgi-type enzymes that cleaving glycoproteins, such as those of subfamily .alpha.-mannosidase II (Man2A1;ManA2). Examples of such enzymes include human enzyme AAC50302, D. melanogaster enzyme (Van den Elsen J. M. et al (2001) EMBO J. 20: 3008-3017), those with the 3D structure according to PDB-reference 1HTY, and others referenced with the catalytic domain in PDB. For cytoplasmic expression, the catalytic domain of the mannosidase is typically fused with an N-terminal targeting peptide (for example as disclosed in the above Section) or expressed with endogenous animal or plant Golgi targeting structures of animal or plant mannosidase II enzymes. After transformation with the catalytic domain of a mannosidase II-type mannosidase, strains are selected that produce GlcNAcMan3 (if GnTI is expressed) or strains are selected that effectively produce GlcNAc2Man3 (if a fusion of GnTI and GnTII is expressed). For strains producing GlcNAcMan3, such strains are further transformed with a polynucleotide encoding a catalytic domain of GnTII and transformant strains that are capable of producing GlcNAc2Man3GlcNAc2 are selected.
[0226] In such embodiment for mimicking the traditional pathway, the filamentous fungal cell is a PMT-deficient filamentous fungal cell as defined in previous sections, and further comprising one or more polynucleotides encoding a polypeptide selected from the group consisting of:
[0227] i) .alpha.1,2 mannosidase,
[0228] ii) N-acetylglucosaminyltransferase I catalytic domain,
[0229] iii) a mannosidase II,
[0230] iv) N-acetylglucosaminyltransferase II catalytic domain,
[0231] v) .beta.1,4 galactosyltransferase, and,
[0232] vi) fucosyltransferase.
[0233] In embodiments using the reduced alg3 pathway, the filamentous fungal cell, such as a Trichoderma cell, has a reduced level of activity of a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase compared to the level of activity in a parent host cell. Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (EC 2.4.1.130) transfers an alpha-D-mannosyl residue from dolichyl-phosphate D-mannose into a membrane lipid-linked oligosaccharide. Typically, the dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase enzyme is encoded by an alg3 gene. In certain embodiments, the filamentous fungal cell for producing glycoproteins with mammalian-like N-glycans has a reduced level of expression of an alg3 gene compared to the level of expression in a parent strain.
[0234] More preferably, the filamentous fungal cell comprises a mutation of alg3. The ALG3 gene may be mutated by any means known in the art, such as point mutations or deletion of the entire alg3 gene. For example, the function of the alg3 protein is reduced or eliminated by the mutation of alg3. In certain embodiments, the alg3 gene is disrupted or deleted from the filamentous fungal cell, such as Trichoderma cell. In certain embodiments, the filamentous fungal cell is a T. reesei cell. SEQ ID NOs: 36 and 37 provide, the nucleic acid and amino acid sequences of the alg3 gene in T. reesei, respectively. In an embodiment the filamentous fungal cell is used for the production of a glycoprotein, wherein the glycan(s) comprise or consist of Man.alpha.3[Man.alpha.6]Man.beta.4GlcNAc.beta.4GlcNAc, and/or a non-reducing end elongated variant thereof.
[0235] In certain embodiments, the filamentous fungal cell has a reduced level of activity of an alpha-1,6-mannosyltransferase compared to the level of activity in a parent strain. Alpha-1,6-mannosyltransferase (EC 2.4.1.232) transfers an alpha-D-mannosyl residue from GDP-mannose into a protein-linked oligosaccharide, forming an elongation initiating alpha-(1->6)-D-mannosyl-D-mannose linkage in the Golgi apparatus. Typically, the alpha-1,6-mannosyltransferase enzyme is encoded by an och1 gene. In certain embodiments, the filamentous fungal cell has a reduced level of expression of an och1 gene compared to the level of expression in a parent filamentous fungal cell. In certain embodiments, the och1 gene is deleted from the filamentous fungal cell.
[0236] The filamentous fungal cells used in the methods of producing glycoprotein with mammalian-like N-glycans may further contain a polynucleotide encoding an N-acetylglucosaminyltransferase I catalytic domain (GnTI) that catalyzes the transfer of N-acetylglucosamine to a terminal Man.alpha.3 and a polynucleotide encoding an N-acetylglucosaminyltransferase II catalytic domain (GnTII), that catalyses N-acetylglucosamine to a terminal Man.alpha.6 residue of an acceptor glycan to produce a complex N-glycan. In one embodiment, said polynucleotides encoding GnTI and GnTII are linked so as to produce a single protein fusion comprising both catalytic domains of GnTI and GnTII.
[0237] As disclosed herein, N-acetylglucosaminyltransferase I (GlcNAc-TI; GnTI; EC 2.4.1.101) catalyzes the reaction UDP-N-acetyl-D-glucosamine+3-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>- ;UDP+3-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl- -R, where R represents the remainder of the N-linked oligosaccharide in the glycan acceptor. An N-acetylglucosaminyltransferase I catalytic domain is any portion of an N-acetylglucosaminyltransferase I enzyme that is capable of catalyzing this reaction. GnTI enzymes are listed in the CAZy database in the glycosyltransferase family 13 (cazy.org/GT13_all). Enzymatically characterized species includes A. thaliana AAR78757.1 (U.S. Pat. No. 6,653,459), C. elegans AAD03023.1 (Chen S. et al J. Biol. Chem 1999; 274(1):288-97), D. melanogaster AAF57454.1 (Sarkar & Schachter Biol Chem. 2001 February; 382(2):209-17); C. griseus AAC52872.1 (Puthalakath H. et al J. Biol. Chem 1996 271(44):27818-22); H. sapiens AAA52563.1 (Kumar R. et al Proc Natl Acad Sci USA. 1990 December; 87(24):9948-52); M. auratus AAD04130.1 (Opat As et al Biochem J. 1998 Dec. 15; 336 (Pt 3):593-8), (including an example of deactivating mutant), Rabbit, O. cuniculus AAA31493.1 (Sarkar M et al. Proc Natl Acad Sci USA. 1991 Jan. 1; 88(1):234-8). Amino acid sequences for N-acetylglucosaminyltransferase I enzymes from various organisms are described for example in PCT/EP2011/070956. Additional examples of characterized active enzymes can be found at cazy.org/GT13_characterized. The 3D structure of the catalytic domain of rabbit GnTI was defined by X-ray crystallography in Unligil U M et al. EMBO J. 2000 Oct. 16; 19(20):5269-80. The Protein Data Bank (PDB) structures for GnTI are 1FO8, 1FO9, 1FOA, 2AM3, 2AM4, 2AM5, and 2APC. In certain embodiments, the N-acetylglucosaminyltransferase I catalytic domain is from the human N-acetylglucosaminyltransferase I enzyme (SEQ ID NO: 38) or variants thereof. In certain embodiments, the N-acetylglucosaminyltransferase I catalytic domain contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acid residues 84-445 of SEQ ID NO: 38. In some embodiments, a shorter sequence can be used as a catalytic domain (e.g. amino acid residues 105-445 of the human enzyme or amino acid residues 107-447 of the rabbit enzyme; Sarkar et al. (1998) Glycoconjugate J 15:193-197). Additional sequences that can be used as the GnTI catalytic domain include amino acid residues from about amino acid 30 to 445 of the human enzyme or any C-terminal stem domain starting between amino acid residue 30 to 105 and continuing to about amino acid 445 of the human enzyme, or corresponding homologous sequence of another GnTI or a catalytically active variant or mutant thereof. The catalytic domain may include N-terminal parts of the enzyme such as all or part of the stem domain, the transmembrane domain, or the cytoplasmic domain.
[0238] As disclosed herein, N-acetylglucosaminyltransferase II (GlcNAc-TII; GnTII; EC 2.4.1.143) catalyzes the reaction UDP-N-acetyl-D-glucosamine+6-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>- ;UDP+6-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl- -R, where R represents the remainder of the N-linked oligosaccharide in the glycan acceptor. An N-acetylglucosaminyltransferase II catalytic domain is any portion of an N-acetylglucosaminyltransferase II enzyme that is capable of catalyzing this reaction. Amino acid sequences for N-acetylglucosaminyltransferase II enzymes from various organisms are listed in WO2012069593. In certain embodiments, the N-acetylglucosaminyltransferase II catalytic domain is from the human N-acetylglucosaminyltransferase II enzyme (SEQ ID NO: 39) or variants thereof. Additional GnTII species are listed in the CAZy database in the glycosyltransferase family 16 (cazy.org/GT16_all). Enzymatically characterized species include GnTII of C. elegans, D. melanogaster, Homo sapiens (NP_002399.1), Rattus norvegicus, Sus scrofa (cazy.org/GT16_characterized). In certain embodiments, the N-acetylglucosaminyltransferase II catalytic domain contains a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acid residues from about 30 to about 447 of SEQ ID NO: 39. The catalytic domain may include N-terminal parts of the enzyme such as all or part of the stem domain, the transmembrane domain, or the cytoplasmic domain.
[0239] In embodiments where the filamentous fungal cell contains a fusion protein of the invention, the fusion protein may further contain a spacer in between the N-acetylglucosaminyltransferase I catalytic domain and the N-acetylglucosaminyltransferase II catalytic domain. In certain embodiments, the spacer is an EGIV spacer, a 2.times.G4S spacer, a 3.times.G4S spacer, or a CBHI spacer. In other embodiments, the spacer contains a sequence from a stem domain.
[0240] For ER/Golgi expression the N-acetylglucosaminyltransferase I and/or N-acetylglucosaminyltransferase II catalytic domain is typically fused with a targeting peptide or a part of an ER or early Golgi protein, or expressed with an endogenous ER targeting structures of an animal or plant N-acetylglucosaminyltransferase enzyme. In certain preferred embodiments, the N-acetylglucosaminyltransferase I and/or N-acetylglucosaminyltransferase II catalytic domain contains any of the targeting peptides of the invention as described in the section entitled "Targeting sequences". Preferably, the targeting peptide is linked to the N-terminal end of the catalytic domain. In some embodiments, the targeting peptide contains any of the stem domains of the invention as described in the section entitled "Targeting sequences". In certain preferred embodiments, the targeting peptide is a Kre2/Mnt1 targeting peptide. In other embodiments, the targeting peptide further contains a transmembrane domain linked to the N-terminal end of the stem domain or a cytoplasmic domain linked to the N-terminal end of the stem domain. In embodiments where the targeting peptide further contains a transmembrane domain, the targeting peptide may further contain a cytoplasmic domain linked to the N-terminal end of the transmembrane domain.
[0241] The filamentous fungal cells may also contain a polynucleotide encoding a UDP-GlcNAc transporter. The polynucleotide encoding the UDP-GlcNAc transporter may be endogenous (i.e., naturally present) in the host cell, or it may be heterologous to the filamentous fungal cell.
[0242] In certain embodiments, the filamentous fungal cell may further contain a polynucleotide encoding a .alpha.-1,2-mannosidase. The polynucleotide encoding the .alpha.-1,2-mannosidase may be endogenous in the host cell, or it may be heterologous to the host cell. Heterologous polynucleotides are especially useful for a host cell expressing high-mannose glycans transferred from the Golgi to the ER without effective exo-.alpha.-2-mannosidase cleavage. The .alpha.-1,2-mannosidase may be a mannosidase I type enzyme belonging to the glycoside hydrolase family 47 (cazy.org/GH47_all.html). In certain embodiments the .alpha.-1,2-mannosidase is an enzyme listed at cazy.org/GH47_characterized.html. In particular, the .alpha.-1,2-mannosidase may be an ER-type enzyme that cleaves glycoproteins such as enzymes in the subfamily of ER .alpha.-mannosidase I EC 3.2.1.113 enzymes. Examples of such enzymes include human .alpha.-2-mannosidase 1B (AAC26169), a combination of mammalian ER mannosidases, or a filamentous fungal enzyme such as .alpha.-1,2-mannosidase (MDS1) (T. reesei AAF34579; Maras M et al J Biotech. 77, 2000, 255, or Trire 45717). For ER expression, the catalytic domain of the mannosidase is typically fused with a targeting peptide, such as HDEL, KDEL, or part of an ER or early Golgi protein, or expressed with an endogenous ER targeting structures of an animal or plant mannosidase I enzyme.
[0243] In certain embodiments, the filamentous fungal cell may also further contain a polynucleotide encoding a galactosyltransferase. Galactosyltransferases transfer .beta.-linked galactosyl residues to terminal N-acetylglucosaminyl residue. In certain embodiments the galactosyltransferase is a .beta.-1,4-galactosyltransferase. Generally, .beta.-1,4-galactosyltransferases belong to the CAZy glycosyltransferase family 7 (cazy.org/GT7_all.html) and include .beta.-N-acetylglucosaminyl-glycopeptide .beta.-1,4-galactosyltransferase (EC 2.4.1.38), which is also known as N-acetylactosamine synthase (EC 2.4.1.90). Useful subfamilies include .beta.4-GalT1, .beta.4-GalT-II, -III, -IV, -V, and -VI, such as mammalian or human .beta.4-GalTI or .beta.4GalT-II, -III, -IV, -V, and -VI or any combinations thereof. .beta.4-GalT1, .beta.4-GalTII, or .beta.4-GalTIII are especially useful for galactosylation of terminal GlcNAc.beta.2-structures on N-glycans such as GlcNAcMan3, GlcNAc2Man3, or GlcNAcMan5 (Guo S. et al. Glycobiology 2001, 11:813-20). The three-dimensional structure of the catalytic region is known (e.g. (2006) J. Mol. Biol. 357: 1619-1633), and the structure has been represented in the PDB database with code 2FYD. The CAZy database includes examples of certain enzymes. Characterized enzymes are also listed in the CAZy database at cazy.org/GT7_characterized.html. Examples of useful .beta.4GalT enzymes include .beta.4GalT1, e.g. bovine Bos taurus enzyme AAA30534.1 (Shaper N. L. et al Proc. Natl. Acad. Sci. U.S.A. 83 (6), 1573-1577 (1986)), human enzyme (Guo S. et al. Glycobiology 2001, 11:813-20), and Mus musculus enzyme AAA37297 (Shaper, N. L. et al. 1998 J. Biol. Chem. 263 (21), 10420-10428); .beta.4GalTII enzymes such as human .beta.4GalTII BAA75819.1, Chinese hamster Cricetulus griseus AAM77195, Mus musculus enzyme BAA34385, and Japanese Medaka fish Oryzias latipes BAH36754; and .beta.4GalTIII enzymes such as human .beta.4GalTIII BAA75820.1, Chinese hamster Cricetulus griseus AAM77196 and Mus musculus enzyme AAF22221.
[0244] The galactosyltransferase may be expressed in the plasma membrane of the host cell. A heterologous targeting peptide, such as a Kre2 peptide described in Schwientek J. Biol. Chem 1996 3398, may be used. Promoters that may be used for expression of the galactosyltransferase include constitutive promoters such as gpd, promoters of endogenous glycosylation enzymes and glycosyltransferases such as mannosyltransferases that synthesize N-glycans in the Golgi or ER, and inducible promoters of high-yield endogenous proteins such as the cbh1 promoter.
[0245] In certain embodiments of the invention where the filamentous fungal cell contains a polynucleotide encoding a galactosyltransferase, the filamentous fungal cell also contains a polynucleotide encoding a UDP-Gal 4 epimerase and/or UDP-Gal transporter. In certain embodiments of the invention where the filamentous fungal cell contains a polynucleotide encoding a galactosyltransferase, lactose may be used as the carbon source instead of glucose when culturing the host cell. The culture medium may be between pH 4.5 and 7.0 or between 5.0 and 6.5. In certain embodiments of the invention where the filamentous fungal cell contains a polynucleotide encoding a galactosyltransferase and a polynucleotide encoding a UDP-Gal 4 epimerase and/or UDP-Gal transporter, a divalent cation such as Mn2+, Ca2+ or Mg2+ may be added to the cell culture medium.
[0246] Accordingly, in certain embodiments, the filamentous fungal cell of the invention, for example, selected among Neurospora, Trichoderma, Myceliophthora or Chrysosporium cell, and more preferably Trichoderma reesei cell, may comprise the following features:
[0247] a) a mutation in at least one endogenous protease that reduces or eliminates the activity of said endogenous protease, preferably the protease activity of two or three or more endogenous proteases is reduced, for example, pep1, tsp1, gap1 and/or slp1 proteases, in order to improve production or stability of a heterologous protein to be produced,
[0248] b) a mutation in a PMT gene, for example T. reesei pmt1 gene, that reduces or eliminates endogenous O-mannosyltransferase activity compared to a parental Trichoderma cell which does not have said second mutation,
[0249] c) a polynucleotide encoding a protein having at least one serine or threonine, preferably a heterologous glycoprotein, such as an immunoglobulin, an antibody, or a protein fusion comprising Fc fragment of an immunoglobulin.
[0250] d) optionally, a deletion or disruption of the alg3 gene,
[0251] e) optionally, a polynucleotide encoding N-acetylglucosaminyltransferase I catalytic domain and a polynucleotide encoding N-acetylglucosaminyltransferase II catalytic domain,
[0252] f) optionally, a polynucleotide encoding .beta.1,4 galactosyltransferase,
[0253] g) optionally, a polynucleotide or polynucleotides encoding UDP-Gal 4 epimerase and/or transporter.
Targeting Sequences
[0254] In certain embodiments, recombinant enzymes, such as .alpha.1,2 mannosidases, GnTI, or other glycosyltransferases introduced into the filamentous fungal cells, include a targeting peptide linked to the catalytic domains. The term "linked" as used herein means that two polymers of amino acid residues in the case of a polypeptide or two polymers of nucleotides in the case of a polynucleotide are either coupled directly adjacent to each other or are within the same polypeptide or polynucleotide but are separated by intervening amino acid residues or nucleotides. A "targeting peptide", as used herein, refers to any number of consecutive amino acid residues of the recombinant protein that are capable of localizing the recombinant protein to the endoplasmic reticulum (ER) or Golgi apparatus (Golgi) within the host cell. The targeting peptide may be N-terminal or C-terminal to the catalytic domains. In certain embodiments, the targeting peptide is N-terminal to the catalytic domains. In certain embodiments, the targeting peptide provides binding to an ER or Golgi component, such as to a mannosidase II enzyme. In other embodiments, the targeting peptide provides direct binding to the ER or Golgi membrane.
[0255] Components of the targeting peptide may come from any enzyme that normally resides in the ER or Golgi apparatus. Such enzymes include mannosidases, mannosyltransferases, glycosyltransferases, Type 2 Golgi proteins, and MNN2, MNN4, MNN6, MNN9, MNN10, MNS1, KRE2, VAN1, and OCH1 enzymes. Such enzymes may come from a yeast or fungal species such as those of Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Chrysosporium, Chrysosporium lucknowense, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. Sequences for such enzymes can be found in the GenBank sequence database.
[0256] In certain embodiments the targeting peptide comes from the same enzyme and organism as one of the catalytic domains of the recombinant protein. For example, if the recombinant protein includes a human GnTII catalytic domain, the targeting peptide of the recombinant protein is from the human GnTII enzyme. In other embodiments, the targeting peptide may come from a different enzyme and/or organism as the catalytic domains of the recombinant protein.
[0257] Examples of various targeting peptides for use in targeting proteins to the ER or Golgi that may be used for targeting the recombinant enzymes, include: Kre2/Mnt1 N-terminal peptide fused to galactosyltransferase (Schwientek, J B C 1996, 3398), HDEL for localization of mannosidase to ER of yeast cells to produce Man5 (Chiba, J B C 1998, 26298-304; Callewaert, FEBS Lett 2001, 173-178), OCH1 targeting peptide fused to GnTI catalytic domain (Yoshida et al, Glycobiology 1999, 53-8), yeast N-terminal peptide of Mns1 fused to .alpha.2-mannosidase (Martinet et al, Biotech Lett 1998, 1171), N-terminal portion of Kre2 linked to catalytic domain of GnTI or .beta.4GalT (Vervecken, Appl. Environ Microb 2004, 2639-46), various approaches reviewed in Wildt and Gerngross (Nature Rev Biotech 2005, 119), full-length GnTI in Aspergillus nidulans (Kalsner et al, Glycocon. J 1995, 360-370), full-length GnTI in Aspergillus oryzae (Kasajima et al, Biosci Biotech Biochem 2006, 2662-8), portion of yeast Sec12 localization structure fused to C. elegans GnTI in Aspergillus (Kainz et al 2008), N-terminal portion of yeast Mnn9 fused to human GnTI in Aspergillus (Kainz et al 2008), N-terminal portion of Aspergillus Mnn10 fused to human GnTI (Kainz et al, Appl. Environ Microb 2008, 1076-86), and full-length human GnTI in T. reesei (Maras et al, FEBS Lett 1999, 365-70).
[0258] In certain embodiments the targeting peptide is an N-terminal portion of the Mnt1/Kre2 targeting peptide having the amino acid sequence of SEQ ID NO: 40 (for example encoded by the polynucleotide of SEQ ID NO:41). In certain embodiments, the targeting peptide is selected from human GNT2, KRE2, KRE2-like, Och1, Anp1, Van1 as shown in the Table 1 below:
TABLE-US-00002 TABLE 1 Amino acid sequence of targeting peptides Protein TreID Amino acid sequence human -- MRFRIYKRKVLILTLVVAACGFVLWSSNGRQR GNT2 KNEALAPPLLDAEPARGAGGRGGDHP (SEQ ID NO: 42) KRE2 21576 MASTNARYVRYLLIAFFTILVFYFVSNSKYEGV DLNKGTFTAPDSTKTTPK (SEQ ID NO: 43) KRE2- 69211 MAIARPVRALGGLAAILWCFFLYQLLRPSSSY like NSPGDRYINFERDPNLDPTG (SEQ ID NO: 44) Ochi 65646 MLNPRRALIAAAFILTVFFLISRSHNSESASTS (SEQ ID NO: 45) Anp1 82551 MMPRHHSSGFSNGYPRADTFEISPHRFQPRA TLPPHRKRKRTAIRVGIAVVVILVLVLWFGQPR SVASLISLGILSGYDDLKLE (SEQ ID NO: 46) Van1 81211 MLLPKGGLDWRSARAQIPPTRALWNAVTRTR FILLVGITGLILLLWRGVSTSASE (SEQ ID NO: 47)
[0259] Further examples of sequences that may be used for targeting peptides include the targeting sequences as described in WO2012/069593.
[0260] Uncharacterized sequences may be tested for use as targeting peptides by expressing enzymes of the glycosylation pathway in a host cell, where one of the enzymes contains the uncharacterized sequence as the sole targeting peptide, and measuring the glycans produced in view of the cytoplasmic localization of glycan biosynthesis (e.g. as in Schwientek J B C 1996 3398), or by expressing a fluorescent reporter protein fused with the targeting peptide, and analysing the localization of the protein in the Golgi by immunofluorescence or by fractionating the cytoplasmic membranes of the Golgi and measuring the location of the protein.
Methods for Producing a Protein Having Reduced O-Mannosylation
[0261] The filamentous fungal cells as described above are useful in methods for producing a protein having reduced O-mannosylation.
[0262] Accordingly, in another aspect, the invention relates to a method for producing a protein having reduced O-mannosylation, comprising:
[0263] a) providing a PMT-deficient Trichoderma cell having a mutation in a PMT gene that reduces endogenous protein O-mannosyltransferase activity as compared to parental strain which does not have such mutation, and further comprising a polynucleotide encoding a protein with serine or threonine, which may be O-mannosylated,
[0264] b) culturing said PMT-deficient Trichoderma cell to produce said protein having reduced O-mannosylation.
[0265] In such method, the produced protein has reduced O-mannosylation due to said mutation in said PMT gene as described in the previous sections. The PMT-deficient Trichoderma cell may optionally have reduced endogenous protease activity as described in the previous sections.
[0266] The filamentous fungal cells and methods of the invention are useful for the production of protein with serine or threonine which may be O-mannosylated. For example, it is particularly useful for the production of protein which are O-mannosylated when produced in a parental PMT-functional filamentous fungal host cell, for example, in at least one Trichoderma cell which is wild type for PMT1 gene, such as SEQ ID NO:1.
[0267] In methods of the invention, certain growth media include, for example, common commercially-prepared media such as Luria-Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular host cell will be known by someone skilled in the art of microbiology or fermentation science. Culture medium typically has the Trichoderma reesei minimal medium (Penttila et al., 1987, Gene 61, 155-164) as a basis, supplemented with substances inducing the production promoter such as lactose, cellulose, spent grain or sophorose. Temperature ranges and other conditions suitable for growth are known in the art (see, e.g., Bailey and Ollis 1986). In certain embodiments the pH of cell culture is between 3.5 and 7.5, between 4.0 and 7.0, between 4.5 and 6.5, between 5 and 5.5, or at 5.5. In certain embodiments, to produce an antibody the filamentous fungal cell or Trichoderma fungal cell is cultured at a pH range selected from 4.7 to 6.5; pH 4.8 to 6.0; pH 4.9 to 5.9; and pH 5.0 to 5.8.
[0268] In some embodiments, the protein which may be O-mannosylated is a heterologous protein, preferably a mammalian protein. In other embodiments, the heterologous protein is a non-mammalian protein.
[0269] In certain embodiments, the protein which may be O-mannosylated is a glycoprotein with N-glycan posttranslational modifications.
[0270] In certain embodiments, a mammalian protein which may be O-mannosylated is selected from an immunoglobulin, immunoglobulin or antibody heavy or light chain, a monoclonal antibody, a Fab fragment, an F(ab')2 antibody fragment, a single chain antibody, a monomeric or multimeric single domain antibody, a camelid antibody, or their antigen-binding fragments.
[0271] A fragment of a protein, as used herein, consists of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 consecutive amino acids of a reference protein.
[0272] As used herein, an "immunoglobulin" refers to a multimeric protein containing a heavy chain and a light chain covalently coupled together and capable of specifically combining with antigen. Immunoglobulin molecules are a large family of molecules that include several types of molecules such as IgM, IgD, IgG, IgA, and IgE.
[0273] As used herein, an "antibody" refers to intact immunoglobulin molecules, as well as fragments thereof which are capable of binding an antigen. These include hybrid (chimeric) antibody molecules (see, e.g., Winter et al. Nature 349:293-99225, 1991; and U.S. Pat. No. 4,816,567 226); F(ab')2 molecules; non-covalent heterodimers; dimeric and trimeric antibody fragment constructs; humanized antibody molecules (see e.g., Riechmann et al. Nature 332, 323-27, 1988; Verhoeyan et al. Science 239, 1534-36, 1988; and GB 2,276,169); and any functional fragments obtained from such molecules, as well as antibodies obtained through non-conventional processes such as phage display or transgenic mice. Preferably, the antibodies are classical antibodies with Fc region. Methods of manufacturing antibodies are well known in the art.
[0274] In further embodiments, the yield of the mammalian glycoprotein is at least 0.5, at least 1, at least 2, at least 3, at least 4, or at least 5 grams per liter.
[0275] In certain embodiments, the mammalian glycoprotein is an antibody, optionally, IgG1, IgG2, IgG3, or IgG4. In further embodiments, the yield of the antibody is at least 0.5, at least 1, at least 2, at least 3, at least 4, or at least 5 grams per liter. In further embodiments, the mammalian glycoprotein is an antibody, and the antibody contains at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of a natural antibody C-terminus and N-terminus without additional amino acid residues. In other embodiments, the mammalian glycoprotein is an antibody, and the antibody contains at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of a natural antibody C-terminus and N-terminus that do not lack any C-terminal or N-terminal amino acid residues.
[0276] In certain embodiments where the mammalian glycoprotein is purified from cell culture, the culture containing the mammalian glycoprotein contains polypeptide fragments that make up a mass percentage that is less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the mass of the produced polypeptides. In certain preferred embodiments, the mammalian glycoprotein is an antibody, and the polypeptide fragments are heavy chain fragments and/or light chain fragments. In other embodiments, where the mammalian glycoprotein is an antibody and the antibody purified from cell culture, the culture containing the antibody contains free heavy chains and/or free light chains that make up a mass percentage that is less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the mass of the produced antibody. Methods of determining the mass percentage of polypeptide fragments are well known in the art and include, measuring signal intensity from an SDS-gel.
[0277] In certain embodiments, where the protein with reduced O-mannosylation, e.g. an antibody, is purified from cell culture, the culture contains at least 70%, 80%, 90%, 95% or 100% of the proteins that is not O-mannosylated (mol %, as determined for example by MALDI TOF MS analysis, and measuring area or intensity of peaks as described in the Example 1 below).
[0278] In certain embodiments where the protein with at least one serine or threonine residue which may be O-mannosylated is purified from cell culture, and where the strain is a Trichoderma cell genetically engineered to produce complex N-glycans, the culture further comprises at least 5%, 10%, 15%, 20%, 25%, 30% of secreted complex neutral N-glycans (mol %) compared to total secreted neutral N-glycans (as measured for example as described in WO2012069593).
[0279] In other embodiments, the heterologous protein with reduced O-mannosylation, for example, the antibody, comprises the trimannosyl N-glycan structure Man.alpha.3[Man.alpha.6]Man.beta.4GlcNAc.beta.4GlcNAc. In some embodiments, the Man.alpha.3[Man.alpha.6]Man.beta.4GlcNAc.beta.4GlcNAc structure represents at least 20%, 30%; 40%, 50%; 60%, 70%, 80% (mol %) or more, of the total N-glycans of the heterologous protein with reduced O-mannosylation. In other embodiments, the heterologous protein with reduced O-mannosylation comprises the G0 N-glycan structure GlcNAc.beta.2Man.alpha.3[GlcNAc.beta.2Man.alpha.6]Man.beta.4GlcNAc.beta.4- GlcNAc. In other embodiments, the non-fucosylated G0 glycoform structure represents at least 20%, 30%; 40%, 50%; 60%, 70%, 80% (mol %) or more, of the total N-glycans of the heterologous protein with reduced O-mannosylation. In other embodiments, galactosylated N-glycans represents less (mol %) than 0.5%, 0.1%, 0.05%, 0.01% of total N-glycans of the culture, and/or of the heterologous protein with reduced O-mannosylation, for example an antibody. In certain embodiments, the culture or the heterologous protein, for example an antibody, comprises no galactosylated N-glycans.
[0280] In certain embodiments, the heterologous (purified) protein is an antibody, a light chain antibody, a heavy chain antibody or a Fab, that comprises Man3, GlcNAcMan3, Man5, GlcNAcMan5, G0, core G0, G1, or G2 N-glycan structure as major glycoform and less than 35%, 20%, 17%, 15%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less than 0.5% of O-mannosylation level (as mole % as determined for example by MALDI TOF MS analysis, and measuring area or intensity of peaks as described in Example 1).
[0281] In a specific embodiment, the invention therefore relates to a method for producing an antibody having reduced O-mannosylation, comprising:
[0282] a. providing a PMT-deficient Trichoderma cell having
[0283] i. a mutation that reduces endogenous protein O-mannosyltransferase activity as compared to parental strain which does not have such mutation and
[0284] ii. a polynucleotide encoding a light chain antibody and a polynucleotide encoding a heavy chain antibody,
[0285] b. culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation.
[0286] In such specific embodiments of the methods related to the production of antibody, at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% of the produced antibody is not O-mannosylated (mol %, as determined for example by MALDI TOF MS analysis, and measuring area or intensity of peaks as described in Example 1.
[0287] In certain embodiments of any of the disclosed methods, the method includes the further step of providing one or more, two or more, three or more, four or more, or five or more protease inhibitors. In certain embodiments, the protease inhibitors are peptides that are co-expressed with the mammalian polypeptide. In other embodiments, the inhibitors inhibit at least two, at least three, or at least four proteases from a protease family selected from aspartic proteases, trypsin-like serine proteases, subtilisin proteases, and glutamic proteases.
[0288] In certain embodiments of any of the disclosed methods, the filamentous fungal cell or Trichoderma fungal cell also contains a carrier protein. As used herein, a "carrier protein" is portion of a protein that is endogenous to and highly secreted by a filamentous fungal cell or Trichoderma fungal cell. Suitable carrier proteins include, without limitation, those of T. reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase II (CeI6A, or CBHII) (see, e.g., Paloheimo et al Appl. Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T. reesei cellobiohydrolase I (CBHI). In some embodiments, the carrier protein is CBH1. In other embodiments, the carrier protein is a truncated T. reesei CBH1 protein that includes the CBH1 core region and part of the CBH1 linker region. In some embodiments, a carrier such as a cellobiohydrolase or its fragment is fused to an antibody light chain and/or an antibody heavy chain. In some embodiments, a carrier-antibody fusion polypeptide comprises a Kex2 cleavage site. In certain embodiments, Kex2, or other carrier cleaving enzyme, is endogenous to a filamentous fungal cell. In certain embodiments, carrier cleaving protease is heterologous to the filamentous fungal cell, for example, another Kex2 protein derived from yeast or a TEV protease. In certain embodiments, carrier cleaving enzyme is overexpressed. In certain embodiments, the carrier consists of about 469 to 478 amino acids of N-terminal part of the T. reesei CBH1 protein GenBank accession No. EGR44817.1.
[0289] In certain embodiments, the filamentous fungal cell of the invention overexpress KEX2 protease. In an embodiment the heterologous protein is expressed as fusion construct comprising an endogenous fungal polypeptide, a protease site such as a Kex2 cleavage site, and the heterologous protein such as an antibody heavy and/or light chain. Useful 2-7 amino acids combinations preceding Kex2 cleavage site have been described, for example, in Mikosch et al. (1996) J. Biotechnol. 52:97-106; Goller et al. (1998) Appl Environ Microbiol. 64:3202-3208; Spencer et al. (1998) Eur. J. Biochem. 258:107-112; Jalving et al. (2000) Appl. Environ. Microbiol. 66:363-368; Ward et al. (2004) Appl. Environ. Microbiol. 70:2567-2576; Ahn et al. (2004) Appl. Microbiol. Biotechnol. 64:833-839; Paloheimo et al. (2007) Appl Environ Microbiol. 73:3215-3224; Paloheimo et al. (2003) Appl Environ Microbiol. 69:7073-7082; and Margolles-Clark et al. (1996) Eur J Biochem. 237:553-560.
[0290] The invention further relates to the protein composition, for example the antibody composition, obtainable or obtained by the method as disclosed above.
[0291] In specific embodiment, such antibody composition obtainable or obtained by the methods of the invention, comprises at least 70%, 80%, 90%, 95%, or 100% of the antibodies that are not O-mannosylated (mol %, as determined for example by MALDI TOF MS analysis, and measuring area or intensity of peaks as described in Example 1). In other specific embodiments, such antibody composition further comprises as 50%, 60%, 70% or 80% (mole % neutral N-glycan), of the following glycoform:
[0292] (i) Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4GlcNA.beta.- 4GlcNAc (Man5 glycoform);
[0293] (ii) GlcNAc.beta.2Man.alpha.3[Man.alpha.6(Man.alpha.3)Man.alpha.6]Man.beta.4Gl- cNA.beta.4GlcNAc, or .beta.4-galactosylated variant thereof;
[0294] (iii) Man.alpha.6(Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc;
[0295] (iv) Man.alpha.6(GlcNAc.beta.2Man.alpha.3)Man.beta.4GlcNA.beta.4GlcNAc, or .beta.4-galactosylated variant thereof: or,
[0296] (v) complex type N-glycans selected from the G0, G1 or G2 glycoform.
[0297] In some embodiments the N-glycan glycoform according to iii-v comprises less than 15%, 10%, 7%, 5%, 3%, 1% or 0.5% or is devoid of Man5 glycan as defined in i) above.
[0298] The invention also relates to a method of reducing O-mannosylation level of a recombinant glycoprotein composition produced in a Trichoderma cell, said method consisting of using a Trichoderma cell having a mutation in a PMT gene wherein said PMT gene is either:
[0299] a. PMT1 gene comprising the polynucleotide of SEQ ID NO:1,
[0300] b. a functional homologous gene of PMT1 gene, which gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or,
[0301] c. a polynucleotide encoding a polypeptide having at least 50%, at least 60%, at least 70%, at least 90%, or at least 95% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.
[0302] In one specific embodiment of such method, said Trichoderma cell is Trichoderma reesei.
[0303] In another specific embodiment of such method, said recombinant glycoprotein comprises at least a light chain antibody or its fragments comprising at least one serine or threonine residue and with at least one N-glycan.
EXAMPLES
[0304] As more specifically exemplified in Example 2, after deletion of pmt1, almost 95% of purified mAb and 70% of Fab molecules no longer contained any O-mannose residues. In contrast, as exemplified in Examples 3 to 4, O-mannosylation level analysis performed on pmt2 and pmt3 deletion strains did not exhibit any appreciable reduction in O-mannosylation. Together with the titer and growth analysis set forth in Example 2, these results demonstrate that filamentous fungal cells, such as Trichoderma cells, can be genetically modified to reduce or suppress O-mannosylation activity, without adversely affecting viability and yield of produced glycoproteins. As such, pmt1 is identified a valuable target to reduce O-mannosylation of secreted proteins and to improve product quality of biopharmaceuticals produced by Trichoderma reesei.
Example 1
Pmt1 Deletion in a Trichoderma reesei Strain
[0305] This example demonstrates that pmt1 is a valuable target to reduce O-mannosylation of secreted proteins and to improve product quality of biopharmaceuticals produced by Trichoderma reesei.
Generation of Pmt1 Deletion Plasmids
[0306] Three different deletion plasmids (pTTv36, pTTv124, pTTv185) were constructed for deletion of the protein O-mannosyltransferase gene pmt1 (TrelD75421). All the plasmids contain the same 5' and 3' flanking regions for correct integration to the pmt1 locus. The difference between the three plasmids is the marker used in the selection; pTTv36 contains a gene encoding acetamidase of Aspergillus nidulans (amdS), pTTv124 contains a loopout version (blaster cassette) of the amdS marker and pTTv185 a loopout version (blaster cassette) of a gene encoding orotidine-5'-monophosphate (OMP) decarboxylase of T. reesei (pyr4).
[0307] The third deletion construct, pTTv185, for the protein O-mannosyltransferase gene pmt1 (TrelD75421) was designed to enable removal of the selection marker from the Trichoderma reesei genome after successful integration and thereby recycling of the selection marker for subsequent transformations. In this approach, the recycling of the marker, i.e. removal of pyr4 gene from the deletion construct, resembles so called blaster cassettes developed for yeasts (Hartl, L. and Seiboth, B., 2005, Curr Genet 48:204-211; and Alani, E. et al., 1987, Genetics 116:541-545). Similar blaster cassettes have also been developed for filamentous fungi including Hypocrea jecorina (anamorph: T. reesei) (Hartl, L. and Seiboth, B., 2005, Curr Genet 48:204-211).
[0308] The TrelD number refers to the identification number of a particular protease gene from the Joint Genome Institute Trichoderma reesei v2.0 genome database. Primers for construction of deletion plasmids were designed either manually or using Primer3 software (Primer3 website, Rozen and Skaletsky (2000) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386).
[0309] The principle of the blaster cassette using pyr4 as the marker gene is as follows: pyr4, encoding orotidine-5'-monophosphate (OMP) decarboxylase of T. reesei (Smith, J. L., et al., 1991, Current Genetics 19:27-33) is needed for uridine synthesis. Strains deficient for OMP decarboxylase activity are unable to grow on minimal medium without uridine supplementation (i.e. are uridine auxotrophs). The utilisation of 5-fluoroorotic acid (5-FOA) in generation of mutant strains lacking OMP decarboxylase activity (pyr4.sup.- strains) is based on the conversion of 5-FOA to a toxic intermediate 5-fluoro-UMP by OMP decarboxylase. Therefore, cells which have a mutated pyr4 gene are resistant to 5-FOA, but in addition are also auxotrophic for uridine. The 5-FOA resistance can in principle result also from a mutation in another gene (pyr2, orotate phosphoribosyltransferase), and therefore the spontaneous mutants obtained with this selection need to be verified for the pyr4.sup.- genotype by complementing the mutant with the pyr4 gene. Once mutated, the pyr4 gene can be used as an auxotrophic selection marker in T. reesei. In our blaster cassette pyr4 is followed by a 310 by direct repeat of pyr4 5' untranslated region (5'UTR) and surrounded by 5' and 3' flanking regions of the gene to be deleted. Integration of the deletion cassette is selected via the pyr4 function. Removal of the pyr4 marker is then forced in the presence of 5-FOA by recombination between the two homologous regions (direct repeat of 5'UTR) resulting in looping out of the selection marker and enabling the utilisation of the same blaster cassette (pyr4 loopout) in successive rounds of gene deletions. After looping out, only the 310 bp sequence of 5'UTR remains in the locus.
[0310] Thus, the pyr4 selection marker and the 5' direct repeat (DR) fragment (310 bp of pyr4 5'UTR) were produced by PCR using plasmid containing a genomic copy of T. reesei pyr4 as a template. Both fragments contained 40 bp overlapping sequences needed to clone the plasmid with the loopout cassette using homologous recombination in yeast (see below). To enable possible additional cloning steps, an AscI digestion site was placed between the pyr4 marker and the 5' direct repeat and NotI sites to surround the complete blaster cassette.
[0311] 1100 bp of 5' and 1000 bp of 3' flanking regions were selected as the basis of the pmt1 deletion plasmids. The flanking region fragments were produced by PCR using a T. reesei wild type strain QM6a (ATCC13631) as the template. For the yeast homologous recombination system used in cloning (see below), overlapping sequences for the vector and the selection marker were placed to the appropriate PCR-primers. To enable marker switch in the construct, NotI restriction sites were introduced between the flanking regions and the selection marker. PmeI restriction sites were placed between the vector and the flanking regions for removal of vector sequence prior to transformation into T. reesei. Vector backbone pRS426 was digested with restriction enzymes (EcoRI and XhoI).
[0312] First deletion plasmid for pmt1 (plasmid pTTv36, Table 2) used amdS, a gene encoding acetamidase of Aspergillus nidulans, as the marker. The marker cassette was digested from an existing plasmid pHHO1 with NotI. All fragments used in cloning were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
[0313] To construct the first deletion plasmid pTTv36, the vector backbone and the appropriate marker and flanking region fragments were transformed into Saccharomyces cerevisiae (strain H3488/FY834). The yeast transformation protocol was based on the method for homologous yeast recombination described in the Neurospora knockouts workshop material of Colot and Collopy, (Dartmouth Neurospora genome protocols website), and the Gietz laboratory protocol (University of Manitoba, Gietz laboratory website). The plasmid DNA from the yeast transformants was rescued by transformation into Escherichia coli. A few clones were cultivated, plasmid DNA was isolated and digested to screen for correct recombination using standard laboratory methods. A few clones with correct insert sizes were sequenced and stored.
[0314] To clone the second pmt1 deletion plasmid (pTTv124, Table 2), the amdS marker was removed from the deletion plasmid pTTv36 with NotI digestion and replaced by another variant of the blaster cassette, amdS loopout cassette containing the amdS selection marker gene, followed by AscI restriction site and a 300 bp direct repeat of amdS 5'UTR. The amdS blaster cassette functions in a similar manner to the pyr4 blaster cassette. The clones containing the amdS blaster cassette are able to grow on acetamide as sole nitrogen source. On medium containing 5-fluoroacetamide (5-FAA) a functional amdS gene will convert 5-FAA to a toxic fluoroacetate and therefore, in the presence of 5-FAA, removal of amdS gene is beneficial to the fungus. Removal of amdS blaster cassette is enhanced via the 300 bp DRs in the cassette like in the pyr4 blaster cassette, which enables the amdS gene to loop out via single crossover between the two DRs. Resulting clones are resistant to 5-FAA and unable to grow on acetamide as the sole nitrogen source.
[0315] The fragments needed for the amdS blaster cassette were produced by PCR using a plasmid p3SR2 (Hynes M. J. et al, 1983, Mol. Cell. Biol. 3:1430-1439) containing a genomic copy of the amdS gene as the template. For the yeast homologous recombination system used in cloning (see above), overlapping sequences were placed to the appropriate PCR-primers. To enable marker switch in the construct, NotI restriction sites were kept between the flanking regions and the blaster cassette. Additional restriction sites FseI and AsiSI were introduced to the 5' end of amdS and an AscI site between amdS and amdS 5'DR. The plasmid pTTv124 was constructed using the yeast recombination system described above. The plasmid DNA from the yeast transformants was rescued by transformation into Escherichia coli. A few clones were cultivated, plasmid DNA was isolated and digested to screen for correct recombination using standard laboratory methods. A few clones with correct insert sizes were sequenced and stored.
[0316] To clone the third pmt1 deletion plasmid (pTTv185, Table 2), the amdS marker was removed from the deletion plasmid pTTv36 with NotI digestion and replaced by the pyr4 blaster cassette described above. The pyr4 blaster cassette was obtained from another plasmid with NotI digestion, ligated to NotI cut pTTv36 and transformed into E. coli using standard laboratory methods. A few transformants were cultivated, plasmid DNA isolated and digested to screen for correct ligation and orientation of the pyr4 blaster cassette using standard laboratory methods. One clone with correct insert size and orientation was sequenced and stored.
[0317] These deletion plasmids for pmt1 (pTTv36, pTTv124 and pTTv185) result in 2465 bp deletion in the pmt1 locus and cover the complete coding sequence of PMT1.
TABLE-US-00003 TABLE 2 Primers for generating deletion plasmids pTTv36, pTTv124 and pTTv185 for protein O-mannosyltransferase 1 (pmt1, TreID75421) Deletion plasmid pTTv36 for pmt1 (TreID75421), vector backbone pRS426 Primer Sequence 75421_5'F CGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGGTTT AAACGCTGCAGGGCGTACAGAACT (SEQ ID NO: 48) 75421_5'R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGTGCGG CCGCGGCTCTAAAATGCTTCACAG (SEQ ID NO: 49) 75421_3'F CGGTTCTCATCTGGGCTTGCTCGGTCCTGGCGTAGATCTAGCGG CCGCACGATGATGATGACAGCCAG (SEQ ID NO: 50) 75421_3'R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGTTT AAACCGTCCAGCTCCCGCAGCGCC (SEQ ID NO: 51) Deletion plasmid pTTv124 for pmt1 (TreID75421), vector backbone pTTv36 T282_75421_amds_5for ATCGCTAACTGCTTTCTCTTCTGTGAAGCATTTTAGAGCCGCGGC CGCGGCCGGCCGCGATCGCCTAGATCTACGCCAGGACCG (SEQ ID NO: 52) T283_amds_3rev_loop CGGTCCTGGCGTAGATCTAGGGCGCGCCACTGGAAACGCAACC CTGAA (SEQ ID NO: 53) T284_amds_loop_5for TTCAGGGTTGCGTTTCCAGTGGCGCGCCCTAGATCTACGCCAGG ACCG (SEQ ID NO: 54) T287_75421_loop_3rev AGCATCATGACCGCCCCCTTCTGGCTGTCATCATCATCGTGCGG CCGCGATTATTGCACAAGCAGCGA (SEQ ID NO: 55) Deletion plasmid pTTv185 for pmt1 (TreID75421), vector backbone pTTv36 Primer Sequence no new primers, pTTv36 digested with NotI and ligated with pyr4- loopout fragment obtained from another plasmid
Generation of Pmt1 Deletion Strains M403, M404, M406 and M407
[0318] To generate a pyr4 negative target strain suitable for the deletion of pmt1 using plasmid pTTv185, the MAB01 antibody producing strain M304 was subjected to selection in the presence of 5-fluoro-orotic acid in order to select for strains containing impaired pyr4 genes. The generation of the strain M304 is described in the International Patent Application No. PCT/EP2013/05012. T. reesei strain M304 comprises MAB01 light chain fused to T. reesei truncated CBH1 carrier with NVISKR Kex2 cleavage sequence, MAB01 heavy chain fused to T. reesei truncated CBH1 carrier with AXE1 [DGETVVKR] Kex2 cleavage sequence, .DELTA.pep1.DELTA.tsp1.DELTA.slp1, and overexpresses T. reesei KEX2.
[0319] Spores of M304 were spread onto minimal medium plates containing 20 g/l glucose, 2 g/l proteose peptone, 5 mM uridine and 1.5 g/l 5-FOA, pH 4.8. Some 5-FOA resistant colonies were streaked after 5-7 days onto plates described above with 1 ml/l Triton X-100 supplementation. A few clones were further purified to single cell clones via consecutive purification platings: a small piece of mycelia was picked to 0.8% NaCl-0.025% Tween 20-20% glycerol, suspended thoroughly by vortexing and filtrated through a cotton-filled pipette tip. Purified clones were sporulated on plates containing 39 g/l potato dextrose agarose. These clones were tested for uridine auxotrophy by plating spores onto minimal medium plates (20 g/l glucose, 1 ml/l Triton X-100, pH 4.8) with and without 5 mM uridine supplementation. No growth was observed on plates without uridine indicating the selected clones were putative pyr4.sup.-. Clones were stored for future use and one of them was designated with strain number M317.
[0320] Pmt1 was deleted from M317 (pyr4.sup.- of the strain M304) using the pmt1 deletion cassette from plasmid pTTv185 described above. To remove the vector sequence, plasmid pTTv185 (.DELTA.pmt1-pyr4) was digested with PmeI+XbaI and the correct fragment was purified from an agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 .mu.g of the pmt1 deletion cassette was used to transform strain M317. Preparation of protoplasts and transformation for pyr4 selection were carried out essentially according to methods in Penttila et al. (1987, Gene 61:155-164) and Gruber et al (1990, Curr. Genet. 18:71-76).
[0321] 100 colonies were picked as selective streaks. 40 transformants were screened by PCR using the primers in Table 3 for the correct integration of the deletion cassette using standard laboratory methods. 12 putative deletion clones were purified to single cell clones. Purified clones were rescreened for integration and for deletion of pmt1 ORF using primers on Table 5. Four clones (in duplicate) were pure disruptants (i.e. no signal with ORF primers).
TABLE-US-00004 TABLE 3 Primers for screening integration of deletion cassette pTTv185 and for deletion of protein O-mannosyltransferase 1 (pmt1, TreID75421) from M317. Primer Sequence T296_75421_5int TATGGCTTTAGATGGGGACA (SEQ ID NO: 56) T027_Pyr4_orf_start_rev TGCGTCGCCGTCTCGCTCCT (SEQ ID NO: 57) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 58) T297_75421_3int CCTGTATCGTCCTGTTCC (SEQ ID NO: 59) T359_pmt1_orf_for GCGCCTGTCGAGTCGGCATT (SEQ ID NO: 60) T360_pmt1_orf_rev CACCGGCCATGCTCTTGCCA (SEQ ID NO: 61) T756_pmt1_orf_for2 CAAGGTGCCCTATGTCGC (SEQ ID NO: 62) T757_pmt1_orf_rev2 GATCGGGTCAGGACGGAA (SEQ ID NO: 63)
[0322] Deletion of pmt1 was verified by Southern analyses. DNA for Southern analyses was extracted with Easy-DNA kit for genomic DNA isolation (Invitrogen) essentially according to the manufacturer's instructions.
[0323] Southern analyses were essentially performed according to the protocol for homologous hybridizations in Sambrook et al. (1989, Molecular Cloning: A laboratory manual. 2.sup.nd Ed., Cold Spring Harbor Laboratory Press) using radioactive labeling (.sup.32P-dCTP) and DecaLabel Plus kit (Fermentas). Southern digestion schemes were designed using Geneious Pro software (Geneious website). Fragments for probes were produced by PCR using the primers listed in Table 4 using a T. reesei wild type strain QM6a (ATCC13631) as the template. PCR products were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
TABLE-US-00005 TABLE 4 Primers for production of probe fragments used in Southern analyses of protein O-mannosyltransferase 1 (pmt1, TreID75421) deletion strains. Primer Sequence T635_pmt1_5f_for AGCCTGTCTGAGGGACGG (SEQ ID NO: 64) T636_pmt1_5f_rev CAAGGTCGAGATTCGGCA (SEQ ID NO: 65) T637_pmt1_3f_for CAGAAGGGGGCGGTCAT (SEQ ID NO: 66) T638_pmt1_3f_rev GTCCCAGCTCCCGCTCT (SEQ ID NO: 67) T359_pmt1_orf_for GCGCCTGTCGAGTCGGCATT (SEQ ID NO: 68) T360_pmt1_orf_rev CACCGGCCATGCTCTTGCCA (SEQ ID NO: 69)
[0324] None of the clones hybridised with pmt1 ORF probe (FIG. 1A) indicating successful deletion of pmt1. Analyses using 5' and 3' flank probes revealed that four of the clones were single integrants (FIGS. 1B and 1C; 26-8A and B, 26-21A and B). Four clones gave additional signals and thus indicated multiple integration of the deletion cassette. Four pure clones (with and without additional copies of the deletion cassette) have been stored for future use (M403; 26-8A, M404; 26-19A, M406; 26-168 and M407; 26-198).
Example 2
Analyses of .DELTA.Pmt1 Strains M403, M404, M406 and M407
[0325] Shake flask cultivation of T. reesei M304 and eight pmt1 deletion strains (26-8A (M403), 26-8B, 26-16A, 26-16B (M406), 26-19A (M404), 26-19B (M407), 26-21A, 26-21B) was carried out in Trichoderma minimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, 9 g/l casamino acids, pH 5.5 at +28.degree. C., 200 rpm. Samples were collected on days 3, 5, 7 and 10 by vacuum filtration. Supernatant samples were stored to -20.degree. C. (antibody and glycan analyses) or used in pH determinations. Mycelia for cell dry weight determinations were rinsed once with DDIW and dried at +100.degree. C. for 20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIW and stored to -20.degree. C.
[0326] O-mannosylation status analysis was performed to shake flask cultivations of T. reesei M304, eight pmt1 disruptants (pTTv185: 26-8A, 26-8B, 26-16A, 26-16B, 26-19A, 26-19B, 26-21A, 26-21B). All were cultivated in TrMM--40 g/l lactose--20 g/l SGE--100 mM PIPPS--9 g/l casamino acids, pH 5.5 at +28.degree. C. and samples were taken on time point days 3, 5, 7 and 10.
[0327] MAB01 antibody from each sample from day 7 was purified from supernatants using Protein G HP MultiTrap 96-well plate (GE Healthcare) according to manufacturer's instructions. The antibody was eluted with 0.1 M citrate buffer, pH 2.6 and neutralized with 2 M Tris, pH 9. The concentration was determined via UV absorbance in spectrophotometer against MAB01 standard curve. For O-mannosylation analysis, 10 .mu.g of protein was incubated in 6 M Guanidinium HCl for 30 minutes at +60.degree. C. after which 5 .mu.l of fresh 0.1 M DTT was added and incubated again as above. The samples were purified using Poros R1 96-well plate and the resulting light chains were analysed using MALDI-TOF MS. All were made as duplicates.
[0328] In flask cultures the O-mannosylation status in pmt1 disruptants was remarkably changed; all .DELTA.pmt1 disruptants looked the same--nearly complete loss of O-mannosylation in MAB01 LC (FIG. 2: Spectra of light chain of flask cultured parental T. reesei strain M317 (pyr4.sup.- of M304) (A) and .DELTA.pmt1 disruptant clone 26-8A (B), day 7).
Fermentation of .DELTA.Pmt1 Strain M403
[0329] Fermentation was carried out with .DELTA.pmt1 strain M403 (clone 26-8A; pTTv185 in M317). Fermentation culture medium contained 30 g/l glucose, 60 g/l lactose, 60 g/l whole spent grain at pH 5.5. Lactose feed was started after glucose exhaustion. Growth temperature was shifted from +28.degree. C. to +22.degree. C. after glucose exhaustion. Samples were collected by vacuum filtration. Supernatant samples were stored to -20.degree. C.
[0330] In FIG. 3 is shown the Western analyses of supernatant samples. MAB01 heavy and light chains were detected from supernatant after day three. Despite the deletion of pmt1, that could also reduce O-mannosylation of the linker and thus aid KEX2 cleavage, substantial amount of light chain remains attached to the carrier in the early days of the fermentation. At later stages, the cleavage is more complete but the yield may be affected by the degradation of the heavy chain. Results on antibody titres (Table 7 below) indicate fairly steady expression between days 7 to 10. In this fermentation the pmt1 deletion strain produced approximately equal antibody levels as the parental strain. Higher titres were obtained when the same strain was fermented using a different fermenter.
[0331] M403 (clone 26-8A) was cultivated in fermenter in TrMM, 30 g/l glucose, 60 g/l lactose, 60 g/l spent grain, pH 5.5 with lactose feed. Samples were harvested on days 2, 3 and 5-11. 0-mannosylation level analysis was performed as to flask cultures. The O-mannosylation status was greatly decreased also in fermenter culture (FIG. 4, Table 5).
[0332] The O-mannosylation level was calculated from average of area and intensity (Table 5). Area (Table 6) seems to give more commonly higher rate of non-O-glycosylated LC than intensity (Table 7). In all time points the O-mannosylation level was below 5%.
TABLE-US-00006 TABLE 5 O-mannosylation status of T. reesei strain M403 (pmt1 deletion strain of MAB01 antibody producing strain, clone 26-8A) from fermenter culture. Percentages calculated from area and intensity of single charged signals. In time point d 9 both samples gave 100% to LC, LC + Hex1 being practically absent. 3 d 5 d 6 d 7 d d 8 d 9 d 10 d 11 Average Average Std Average Std Average Std Average Average Average Std Average Std LC 95.8 96.8 0.30 97.5 0.29 97.4 0.36 97.3 100.0 96.6 0.2 95.5 0.11 LC + Hex 4.2 3.2 0.30 2.5 0.29 2.6 0.36 2.7 0.0 3.4 0.2 4.5 0.11
TABLE-US-00007 TABLE 6 The percentages of area values of three parallel samples from fermenter cultured M403 from day 7. Area average Std LC 98.5 0.15 LC + Hex 1.5 0.15
TABLE-US-00008 TABLE 7 The percentages of intensity values of three parallel samples from fermenter cultured M403 from day 7. Intensity average Std LC 96.3 0.57 LC + Hex 3.7 0.57
[0333] No negative effects of strain growth characteristic and secretion capacity were observed. The strain M403 grew well and produced increased amount of antibody in function of time in fermenter culture. The best titer was obtained from day 10 (Table 8). On day 11 the titer is decreased.
TABLE-US-00009 TABLE 8 Titers from fermenter cultured MAB01 producing strain M403. The antibody was purified using Protein G 96-well plate. Time point Days cultured Titer g/l 54:30 hours 2 0.04 71:50 hours 3 0.04 77:45 hours 3 0.07 126:20 hours 5 0.91 148:20 hours 6 1.23 168:20 hours 7 1.47 192:00 hours 8 1.50 217:15 hours 9 1.35 241:00 hours 10 1.52 275:20 hours 11 1.06
[0334] Deletion of pmt1 diminished dramatically MAB01 0-mannosylation; the amount of O-mannosylated LC was .about.61% in parental strain, 3% in the best .DELTA.pmt1 clone in shake flask culture and practically 0% in fermenter culture in time point day 9.
Deletion of Pmt1 in a Fab Expressing Trichoderma reesei Strain
[0335] The pmt1 disruption cassette (pmt1 amdS) was released from its backbone vector pTTv124 described above by restriction digestion and purified through gel extraction. Using protoplast transformation the deletion cassette was introduced to T. reesei strains M304 (3-fold protease deletion strain expressing MAB01) and M307 (4-fold protease deletion strain .DELTA.pep1 .DELTA.tsp1 .DELTA.slp1 .DELTA.gap1, also described in PCT/EP2013/050126 that has been transformed to express a Fab). Transformants were plated to acetamidase selective medium (minimal medium containing acetamide as the sole carbon source).
[0336] Transformants were screened by PCR for homologous integration of the acetamidase marker to the pmt1 locus using a forward primer outside the 5' flanking region fragment of the construct and the reverse primer inside the AmdS selection marker (5' integration) as well as a forward primer inside the AmdS selection marker and a reverse primer outside the 3' flanking region fragment (3' integration). Three independent transformants of each transformation (MAB01 and Fab expressing strains), which gave PCR results displaying correct integration of the construct to the pmt1 locus were selected for single spore purification to obtain uninuclear clones. Proper integration of the disruption cassette was reconfirmed by PCR using the same primer combinations as described above and the absence of the pmt1 gene was verified by using a primer combination targeted to the pmt1 open reading frame. Correct integration of the disruption cassette was additionally verified for all clones applying Southern hybridization. Digested genomic DNA of the three clones as well as the parental strain were probed against the 5' and 3' flanks of the pmt1 gene to confirm modification of the pmt1 locus as expected. Furthermore, the blotted DNA was hybridized with a probe specific to the pmt1 open reading frame in order to substantiate the absence of pmt1.
MAB01 and Fab Expression for O-Mannosylation Analysis
[0337] To evaluate the impact of pmt1 deletion on O-mannosylation levels of mAb and Fab molecules, strains were grown in batch fermentations for 7 days, in media containing 2% yeast extract, 4% cellulose, 4% cellobiose, 2% sorbose, 5 g/L KH2PO4, and 5 g/L (NH4)2SO4. Culture pH was controlled at pH 5.5 (adjusted with NH4OH). The starting temperature was 30.degree. C., which was shifted to 22.degree. C. after 48 hours. mAb fermentations (strains M304, M403, M406 and M407) were carried out in 4 parallel 2 L glas reactor vessels (DASGIP) with a culture volume of 1 L and the Fab fermentation (TR090#5) was done in a 15 L steel tank reactor (Infors) with a culture volume of 6 L. Fab strains (TR090#5, TR090#3, TR090#17) were additionally cultured in shake flasks for 4 days at 28.degree. C. Main media components were 1% yeast extract, 2% cellobiose, 1% sorbose, 15 g/L KH2PO4 and 5 g/L (NH4)2SO4 and the pH was uncontrolled (pH drops from 5.5 to <3 during a time course of cultivation). Culture supernatant samples were taken during the course of the runs and stored at -20.degree. C. Samples were collected daily from the whole course of these cultivations, and production levels were analyzed by affinity liquid chromatography. Samples with maximum production levels were subject to purification and further O-mannosylation analysis.
Analysis of O-mannosylation on Fab and mAb
[0338] O-mannosylation was analyzed on mAb and Fab molecules expressed from both, the pmt1 deletion and parental strains. The mAb and Fab was purified from culture supernatants using Lambda Select Sure and CaptureSelect Fab Lambda (BAC) affinity chromatography resin, respectively, applying conditions as described by the manufactures protocols. Both purified molecules including, the purified mAb and Fab were subjected to RP-LC-QTOF-MS either as intact and/or reduced/alkylated samples.
[0339] For intact analysis, an equivalent of 20 .mu.g protein was injected onto the column. For reduced/alkylated analyses of mAb, an equivalent of 100 .mu.g protein was deglycosylated using PNGase-F enzyme, reduced using DTT and alkylated using iodoacetamide prior to LC-MS analysis. For reduced/alkylated analyses of Fab, an equivalent of 100 .mu.g protein was reduced with DTT and alkylated with iodoacetamide prior to LC-MS analysis. 6 .mu.g of the reduced/alkylated sample were injected onto the column. Reversed-phase chromatography separation was carried out on a 2.1.times.150 mm Zorbax C3 column packed with 5 .mu.m particles, 300 .ANG. pore size the eluents were: eluent A 0.1% TFA in water and eluent B 0.1% TFA in 70% IPA, 20% ACN, 10% water. The column was heated at 75.degree. C. and the flo rate was 200 .mu.L/min. The gradient used for the sample separation is shown in Table 9.
TABLE-US-00010 TABLE 9 HPLC gradient used for intact and reduced/alkylated samples Time % B Flow (mL/min) 0 10 0.1 0.1 10 0.2 2 10 0.2 4 28 0.2 30 36.4 0.2 31 100 0.2 34 100 0.2 35 10 0.2 40 10 0.2
[0340] The HPLC was directly coupled with a Q-TOF Ultima mass spectrometer (Waters, Manchester, UK). The ESI-TOF mass spectrometer was set to run in positive ion mode. The data evaluation of intact and reduced/alkylated analyses was performed using MassLynx analysis software (Waters, Manchester, UK). The deconvolution of the averaged mass spectra from the main UV signals was carried out using the MaxEnt algorithm, a part of the MassLynx analysis software (Waters, Manchester, UK). The deconvolution parameters were the following: "max numbers of iterations" are 8; resolution is 0.1 Da/channel; Uniform Gaussian--width at half height is 1 Da for intact and 0.5 for the reduced chains and minimum intensity ratios are left 30% and right 30%. The estimated level of O-mannosylation (%) was determined using the peak signal height after deconvolution. The observed O-mannosylation levels (%) of mAbs and Fabs from independent pmt1 deletion strains are compared to the ones of the respective parental wild-type strains in Tables 10 and 11.
TABLE-US-00011 TABLE 10 O-mannosylation level [%] of Fabs from different strains Strain Parental Sample M307 TR090#5 TR090#3 TR090#17 Intact Fab 70.1 34.2 34.3 34.7 LC 58.8 10.4 10.1 10.8 HC 42.9 26.1 25.9 25.8
TABLE-US-00012 TABLE 11 O-mannosylation level [%] of MAB01 from different pmt1 deficient strains M403, M406 and M407. Parental strain is M304 Strain in yeast extract medium Sample Parental M403 M406 M407 LC 50.7 5.7 5.8 5.8 HC 4.8 Not Not Not detected detected detected
[0341] The O-mannosylation level was found to be 70% on intact Fab derived from the parental strain and reduced to .about.34% in all three pmt1 deletion strains. The transfer of mannoses was more efficiently diminished on the Fab light chains (10% of residual O-mannosylation on light chains obtained from pmt1 deletion strains vs. 59% for the parental strain), as compared to the heavy chains, for which it decreased from 43% to .about.26%.
[0342] The O-mannosylation level was found to be 50% on the light chain of mAb derived from parental strains and reduced to 5.7-5.8% in all three pmt1 deletion strains. The O-mannosylation level was found to be 4.8% on the heavy chain of mAb derived from parental strains and was completely reduced (below the limit of detection by LC-MS) in all three pmt1 deletion strains.
[0343] In conclusion, after deletion of pmt1, almost 95% of purified mAb and 70% of Fab molecules did no longer contain any O-mannose residues. Therefore, pmt1 is a valuable target to reduce O-mannosylation of secreted proteins and to improve product quality of biopharmaceuticals produced by Trichoderma reesei.
Example 3
Pmt2 Deletion in a Trichoderma reesei Strain
Generation of Pmt2 Deletion Plasmids
[0344] Three different deletion plasmids (pTTv34, pTTv122, pTTv186) were constructed for deletion of the protein O-mannosyltransferase gene pmt2 (TrelD22005). All the plasmids contain the same 5' and 3' flanking regions for correct integration to the pmt2 locus. The difference between the three plasmids is the marker used in the selection; pTTv34 contains a gene encoding acetamidase of Aspergillus nidulans (amdS), pTTv122 contains a loopout version (blaster cassette) of the amdS marker and pTTv186 a loopout version (blaster cassette) of a gene encoding orotidine-5'-monophosphate (OMP) decarboxylase of T. reesei (pyr4).
[0345] 1100 bp of 5' and 1000 bp of 3' flanking regions were selected as the basis of the second protein O-mannosyltransferase gene, pmt2 (TrelD22005), deletion plasmids. The construction of the first plasmid for this gene was carried out essentially as described for pmt1 in Example 1. As for pmt1, the first deletion plasmid for pmt2 (plasmid pTTv34, Table 12) used amdS, a gene encoding acetamidase of Aspergillus nidulans, as the selection marker.
[0346] Like for pmt1 in Example 1, to clone the second deletion plasmid, pTTv122 (Table 12), the amdS marker was removed from the deletion plasmid pTTv34 with NotI digestion and replaced by amdS blaster cassette for which the fragments were produced by PCR (see Example 1 above for details). The plasmid pTTv122 was constructed using the yeast recombination system described in Example 1. The plasmid DNA from the yeast transformants was rescued by transformation into Escherichia coli. A few clones were cultivated, plasmid DNA was isolated and digested to screen for correct recombination using standard laboratory methods. A few clones with correct insert sizes were sequenced and stored.
[0347] The third deletion plasmid for pmt2, pTTv186 (Table 12) was cloned like the third plasmid for pmt1; the amdS blaster cassette was removed from the deletion plasmid pTTv122 with NotI digestion and replaced by the pyr4 blaster cassette described in Example 1. The pyr4 blaster cassette was obtained from another plasmid with NotI digestion, ligated to NotI cut pTTv122 and transformed into E. coli using standard laboratory methods. A few transformants were cultivated, plasmid DNA isolated and digested to screen for correct ligation and orientation of the pyr4 blaster cassette using standard laboratory methods. One clone with correct insert size and orientation was sequenced and stored. These deletion plasmids for pmt2 (pTTv34, pTTv122 and pTTv186, Table 12) result in 3186 bp deletion in the pmt2 locus and cover the complete coding sequence of PMT2.
TABLE-US-00013 TABLE 12 Primers for generating deletion plasmids pTTv34, pTTv122 and pTTv186 for protein O-mannosyltransferase 2 (pmt2, TreID22005). Deletion plasmid pTTv34 for pmt2 (TreID22005), vector backbone pRS426 Primer Sequence 22005_5'F CGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGGT TTAAACGTTTCAGGTACCAACACCTG (SEQ ID NO: 70) 22005_5'R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGTGC GGCCGCGGCGAAGAGTCTGGCGGGGA (SEQ ID NO: 71) 22005_3'F CGGTTCTCATCTGGGCTTGCTCGGTCCTGGCGTAGATCTAGCG GCCGCAAGAGGATGGGGGTAAAGCT (SEQ ID NO: 72) 22005_3'R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGT TTAAACGAGGAGGACTCGTGAGTTAT (SEQ ID NO: 73) Deletion plasmid pTTv122 for pmt2 (TreID22005), vector backbone pTTv34 T280_22005_amds_5for GCGCCCTTCCGCCTCGACAATCCCCGCCAGACTCTTCGCCGC GGCCGCGGCCGGCCGCGATCGCCTAGATCTACGCCAGGACC G (SEQ ID NO: 74) T283_amds_3rev_loop CGGTCCTGGCGTAGATCTAGGGCGCGCCACTGGAAACGCAAC CCTGAA (SEQ ID NO: 75) T284_amds_loop_5for TTCAGGGTTGCGTTTCCAGTGGCGCGCCCTAGATCTACGCCAG GACCG (SEQ ID NO: 76) T285_22005_loop_3rev GAGCTGGCCAGAAAAGACCAAGCTTTACCCCCATCCTCTTGCG GCCGCGATTATTGCACAAGCAGCGA (SEQ ID NO: 77) Deletion plasmid pTTv186 for pmt2 (TreID22005), vector backbone pTTv122 Primer Sequence no new primers, pTTv122 digested with NotI and ligated with pyr4- loopout fragment from another plasmid
Generation of Pmt2 Deletion Strains M338, M339 and M340
[0348] To remove vector sequence plasmid pTTv122 (.DELTA.pmt2-amdS) was digested with PmeI+XbaI and the 5.2 kb fragment purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 .mu.g of the pmt2 deletion cassette was used to transform the strain M124 (M124 strain is described in WO2012/069593). Protoplast preparation and transformation were carried out essentially according to Penttila et al., 1987, Gene 61:155-164 and Gruber et al, 1990, Current Genetics 18:71-76 for amdS selection.
[0349] 120 colonies were picked as selective streaks. 10 transformants were screened by PCR using the primers in Table 13 for the correct integration of the deletion cassette using standard laboratory methods. Five putative deletion clones were purified to single cell clones. Purified clones (two parallel from each) were rescreened for correct integration and for deletion of pmt2 ORF (primers on Table 13). Five clones were selected for Southern analyses.
TABLE-US-00014 TABLE 13 Primers for screening integration of deletion cassette pTTv122 and for deletion of protein O- mannosyltransferase 2 (pmt2, TreID22005) from M124. Primer Sequence T288_22005_5int ACGAGTTGTTTCGTGTACCG (SEQ ID NO: 78) T020_Amds_rev2 CTTTCCATTCATCAGGGATGG (SEQ ID NO: 79) T021_amds_end_fwd GGAGACTCAGTGAAGAGAGG (SEQ ID NO: 80) T289_22005_3int ATGTTGCAGTTGCGAAAG (SEQ ID NO: 81) T290_22005_5orf CCCTCGTCGCAGAAAAGATG (SEQ ID NO: 82) T291_22005_3orf AGCCTCCTTGGGAACCTCAG (SEQ ID NO: 83)
[0350] Deletion of pmt2 was verified by Southern analyses. DNA for Southern analyses was extracted with Easy-DNA kit for genomic DNA isolation (Invitrogen) essentially according to the manufacturer's instructions.
[0351] Southern analyses were essentially performed as described in Example 1. Fragments for probes were produced by PCR using the primers listed in Table 14 using a T. reesei strain M124 as the template for the ORF probe and plasmid pTTv122 for the 5' and 3' flank probes. PCR products were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
TABLE-US-00015 TABLE 14 Primers for production of probe fragments used in Southern analyses of protein O-mannosyltransferase 2 (pmt2, TreID22005) deletion strains. Primer Sequence T639_22005 5' flank CTTAGTGCGGCTGGAGGGCG probe F (SEQ ID NO: 84) T640_22005 5' flank GGCCGGTTCGTGCAACTGGA probe R (SEQ ID NO: 85) T641_22005 3' flank GGCCGCAAGAGGATGGGGGT probe F (SEQ ID NO: 86) T642_22005 3' flank TCGGGCCAGCTGAAGCACAAC probe R (SEQ ID NO: 87) T643_22005 orf 5' probe TTGAGGAACGGCTGCCTGCG (SEQ ID NO: 88) T644_22005 orf 3' probe CGATGGCTCCGTCATCCGCC (SEQ ID NO: 89)
[0352] Three of the clones did not hybridise with pmt2 ORF probe (Data not shown) indicating successful deletion of pmt2. Analyses using 5' and 3' flank probes revealed that the same three clones were single integrants (Data not shown). The two other clones (19-35A and 19-40B) gave signals corresponding to parental strain M124. Three pure clones have been stored for future use (M338; 19-7B, M339; 19-22B and M340; 19-39B).
Analyses of .DELTA.Pmt2 Strains M338, M339 and M340
[0353] Shake flask cultivation of T. reesei strain M124 and the pmt2 deletion strains (19-7B/M338, 19-22B/M339 and 19-39B/M340) was carried out in Trichoderma minimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, pH 5.5 with and without 1 M sorbitol as osmotic stabiliser at +28.degree. C., 200 rpm. Samples were collected on days 3, 5 and 7 by vacuum filtration. Supernatant samples were stored to -20.degree. C. (antibody and glycan analyses) or used in pH determinations. Mycelia for cell dry weight determinations were rinsed once with DDIW and dried at +100.degree. C. for 20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIW and stored to -20.degree. C.
Generation of Pmt2 Deletion Strains M452, M453 and M454
[0354] Generation of M317 is described in Example 1 above.
[0355] To remove vector sequence plasmid pTTv186 (.DELTA.pmt2-pyr4) was digested with PmeI+XbaI and the 4.1 kb fragment purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 .mu.g of the pmt2 deletion cassette was used to transform M317.
[0356] Protoplast preparation and transformation were carried out essentially according to Penttila et al., 1987, Gene 61:155-164 and Gruber et al, 1990, Current Genetics 18:71-76 for pyr4 selection.
[0357] 100 colonies were picked as selective streaks. 20 transformants were screened by PCR using the primers in Table 15 for the correct integration of the deletion cassette using standard laboratory methods. Nine putative deletion clones were purified to single cell clones. Purified clones were rescreened for 5' integration and for deletion of pmt2 ORF (primers on Table 14). Three clones were pure deletants (i.e. no signal with ORF primers).
TABLE-US-00016 TABLE 15 Primers for screening integration of deletion cassette pTTv186 and for deletion of protein O-mannosyltransferase 2 (pmt2, TreID22005) from M317. Primer Sequence T288_22005_5int ACGAGTTGTTTCGTGTACCG (SEQ ID NO: 90) T027_Pyr4_orf_start_rev TGCGTCGCCGTCTCGCTCCT (SEQ ID NO: 91) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 92) T289_22005_3int ATGTTGCAGTTGCGAAAG (SEQ ID NO: 93) T290_22005_5orf CCCTCGTCGCAGAAAAGATG (SEQ ID NO: 94) T291_22005_3orf AGCCTCCTTGGGAACCTCAG (SEQ ID NO: 95)
[0358] Deletion of pmt2 was verified by Southern analyses. DNA for Southern analyses was extracted with Easy-DNA kit for genomic DNA isolation (Invitrogen) essentially according to the manufacturer's instructions.
[0359] Southern analyses were essentially performed as described in Example 1. Fragments for probes were produced by PCR using the primers listed in Table 16 using a T. reesei wild type strain QM6a (ATCC13631) as the template for pmt2 ORF probe and plasmid pTTv186 for 5' and 3' flank probes. PCR products were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
TABLE-US-00017 TABLE 16 Primers for production of probe fragments used in Southern analyses of protein O-mannosyltransferase 2 (pmt2, TreID22005) deletion clones. Primer Sequence T639_22005 5' flank CTTAGTGCGGCTGGAGGGCG probe F (SEQ ID NO: 96) T640_22005 5' flank GGCCGGTTCGTGCAACTGGA probe R (SEQ ID NO: 97) T641_22005 3' flank GGCCGCAAGAGGATGGGGGT probe F (SEQ ID NO: 98) T642_22005 3' flank TCGGGCCAGCTGAAGCACAAC probe R (SEQ ID NO: 99) T290_22005_5orf CCCTCGTCGCAGAAAAGATG (SEQ ID NO: 100) T291_22005_3orf AGCCTCCTTGGGAACCTCAG (SEQ ID NO: 101)
[0360] None of the clones hybridised with pmt2 ORF probe (Data not shown) indicating successful deletion of pmt2. Analyses using 5' and 3' flank probes revealed that two of the clones were single integrants (Data not shown). One clone gave additional signal from the 3'flank probing (Data not shown) and thus indicated partial or multiple integration of the deletion cassette. Three pure clones (with and without additional copies of the deletion cassette) have been stored for future use (M452; 27-10A, M453; 27-17A and M454: 27-18B).
Analyses of .DELTA.Pmt2 Strains M452, M453 and M454
[0361] Shake flask cultivation of T. reesei strain M304 and three pmt2 deletion strains (27-10A/M452, 27-17A/M453 and 27-18B/M454) was carried out in Trichoderma minimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, 9 g/l casamino acids, pH 5.5 at +28.degree. C., 200 rpm. Samples were collected on days 3, 5, 7 and 10 by vacuum filtration. Supernatant samples were stored to -20.degree. C. (antibody and glycan analyses) or used in pH determinations. Mycelia for cell dry weight determinations were rinsed once with DDIW and dried at +100.degree. C. for 20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIW and stored to -20.degree. C.
[0362] O-mannosylation level analysis was performed to pmt2 deletion strains as to flask cultures of pmt1 deletion strains. No difference was observed in O-mannosylation compared to parental strain M304.
Example 4
Pmt3 Deletion in a Trichoderma reesei Strain
Generation of Pmt3 Deletion Plasmids
[0363] Three different deletion plasmids (pTTv35, pTTv123, pTTv187) were constructed for deletion of the protein O-mannosyltransferase gene pmt3 (TrelD22527). All the plasmids contain the same 5' and 3' flanking regions for correct integration to the pmt3 locus. The difference between the three plasmids is the marker used in the selection; pTTv35 contains a gene encoding acetamidase of Aspergillus nidulans (amdS), pTTv123 contains a loopout version (blaster cassette) of the amdS marker and pTTv187 a loopout version (blaster cassette) of a gene encoding orotidine-5'-monophosphate (OMP) decarboxylase of T. reesei (pyr4).
[0364] 1100 bp of 5' and 1000 bp of 3' flanking regions were selected as the basis of the third protein O-mannosyltransferase gene, pmt3 (TrelD22527), deletion plasmids. The construction of the first plasmid for this gene was carried out essentially as described for pmt1 in Example 1. As for pmt1, the first deletion plasmid for pmt3 (plasmid pTTv35, Table 17) used amdS, a gene encoding acetamidase of Aspergillus nidulans, as the selection marker.
[0365] Like for pmt1 in Example 1, to clone the second deletion plasmid, pTTv123 (Table 16), the amdS marker was removed from the deletion plasmid pTTv35 with NotI digestion and replaced by amdS blaster cassette for which the fragments were produced by PCR (see Example 1 above for details). The plasmid pTTv123 was constructed using the yeast recombination system described in Example 1. The plasmid DNA from the yeast transformants was rescued by transformation into Escherichia coli. A few clones were cultivated, plasmid DNA was isolated and digested to screen for correct recombination using standard laboratory methods. A few clones with correct insert sizes were sequenced and stored.
[0366] The third deletion plasmid for pmt3, pTTv187 (Table 17) was cloned like the third plasmid for pmt1; the amdS blaster cassette was removed from the deletion plasmid pTTv123 with NotI digestion and replaced by the pyr4 blaster cassette described in Example 1. The pyr4 blaster cassette was obtained from another plasmid with NotI digestion, ligated to NotI cut pTTv123 and transformed into E. coli using standard laboratory methods. A few transformants were cultivated, plasmid DNA isolated and digested to screen for correct ligation and orientation of the pyr4 blaster cassette using standard laboratory methods. One clone with correct insert size and orientation was sequenced and stored. These deletion plasmids for pmt3 (pTTv35, pTTv123 and pTTv187, Table 17) result in 2495 bp deletion in the pmt3 locus and cover the complete coding sequence of PMT3.
TABLE-US-00018 TABLE 17 Primers for generating deletion plasmids pTTv35, pTTv123 and pTTv187 for protein O-mannosyltransferase 3 (pmt3, TreID22527). Deletion plasmid pTTv35 for pmt3 (TreID22527), vector backbone pRS426 Primer Sequence 22527_5'F CGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGAC GGTTTAAACGTGTTTAAATTTGATGAGGC (SEQ ID NO: 102) 22527_5'R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGT GCGGCCGCGGTCTCAGAGACAGCCTTCT (SEQ ID NO: 103) 22527_3'F CGGTTCTCATCTGGGCTTGCTCGGTCCTGGCGTAGATCTA GCGGCCGCACTCGGCTTCTTTGTCCGAG (SEQ ID NO: 104) 22527_3'R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG CGTTTAAACTCCTCGTCGGCAACAAGGCC (SEQ ID NO: 105) Deletion plasmid pTTv123 for pmt3 (TreID22527), vector backbone pTTv35 T281_22527_amds_5for GCAGATCTGGGGGAGGAATCAGAAGGCTGTCTCTGAGACC GCGGCCGCGGCCGGCCGCGATCGCCTAGATCTACGCCAG GACCG (SEQ ID NO: 106) T283_amds_3rev_loop CGGTCCTGGCGTAGATCTAGGGCGCGCCACTGGAAACGC AACCCTGAA (SEQ ID NO: 107) T284_amds_loop_5for TTCAGGGTTGCGTTTCCAGTGGCGCGCCCTAGATCTACGC CAGGACCG (SEQ ID NO: 108) T286_22527_loop_3rev AAAGTGGGCGAGCTGAGATACTCGGACAAAGAAGCCGAGT GCGGCCGCGATTATTGCACAAGCAGCGA (SEQ ID NO: 109) Deletion plasmid pTTv187 for pmt3 (TreID22527), vector backbone pTTv123 Primer Sequence no new primers, pTTv123 digested with NotI and ligated with pyr4- loopout fragment from another plasmid.
Generation of Pmt3 Deletion Strains M341 and M342
[0367] To remove vector sequence plasmid pTTv123 (.DELTA.pmt3-amdS) was digested with PmeI+XbaI and the 5.2 kb fragment purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 .mu.g of the pmt3 deletion cassette was used to transform the strain M124. Protoplast preparation and transformation were carried out essentially according to Penttila et al., 1987, Gene 61:155-164 and Gruber et al, 1990, Current Genetics 18:71-76 for amdS selection.
[0368] 120 colonies were picked as selective streaks. 10 transformants were screened by PCR using the primers in Table 18 for the correct integration of the deletion cassette using standard laboratory methods. Three putative deletion clones were purified to single cell clones. Purified clones (three parallel from each) were rescreened for correct integration and for deletion of pmt3 ORF (primers on Table 18). Three clones were selected for Southern analyses.
TABLE-US-00019 TABLE 18 Primers for screening integration of deletion cassette pTTv123 and for deletion of protein O- mannosyltransferase 3 (pmt3, TreID22527) from M124. Primer Sequence T292_22527_5int ACGGGAGATCTCGGAAAA (SEQ ID NO: 110) T020_Amds_rev2 CTTTCCATTCATCAGGGATGG (SEQ ID NO: 111) T021_amds_end_fwd GGAGACTCAGTGAAGAGAGG (SEQ ID NO: 112) T293_22527_3int ATGAAGCTCAGCCTGTGG (SEQ ID NO: 113) T294_22527_5orf GGGGACGGCTTGAGGAAG (SEQ ID NO: 114) T295_22527_3orf CTGCTTGCTGCTTCCAGTCA (SEQ ID NO: 115)
[0369] Deletion of pmt3 was verified by Southern analyses. DNA for Southern analyses was extracted with Easy-DNA kit for genomic DNA isolation (Invitrogen) essentially according to the manufacturer's instructions.
[0370] Southern analyses were essentially performed as described in Example 1. Fragments for probes were produced by PCR using the primers listed in Table 19 using a T. reesei strain M124 as the template for the ORF probe and plasmid pTTv123 for the 5' and 3' flank probes. PCR products were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
TABLE-US-00020 TABLE 19 Primers for production of probe fragments used in Southern analyses of protein O- mannosyltransferase 3 (pmt3, TreID22527) deletion strains. Primer Sequence T645_22527 5' flank TGGCAGATGCCGAAAGGCGG probe F (SEQ ID NO: 116) T646_22527 5' flank TGGCAACCAGCTGTGGCTCC probe R (SEQ ID NO: 117) T647_22527 3' flank CGGCCGCACTCGGCTTCTTT probe F (SEQ ID NO: 118) T648_22527 3' flank GAGTGGGCTAGGCGCAACGG probe R (SEQ ID NO: 119) T649_22527 orf 5' GGATCGGCCACTGCCACCAC probe (SEQ ID NO: 120) T650_22527 orf 3' GCCCACTTCTCTGCGCGTGT probe (SEQ ID NO: 121)
[0371] Two of the clones did not hybridise with pmt3 ORF probe (Data not shown) indicating successful deletion of pmt3. Analyses using 5' and 3' flank probes revealed that the same two clones were single integrants (Data not shown). One clone (20-32C) gave signals corresponding to parental strain M124. Two clones have been stored for future use (M341; 20-34C and M342; 20-35B).
Analyses of .DELTA.Pmt3 Strains M341 and M342
[0372] Shake flask cultivation of T. reesei strain M124 and the pmt3 deletion strains (20-34C/M341 and 20-35B/M342) was carried out in Trichoderma minimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, pH 5.5 with and without 1 M sorbitol as osmotic stabiliser at +28.degree. C., 200 rpm. Samples were collected on days 3, 5 and 7 by vacuum filtration. Supernatant samples were stored to -20.degree. C. (antibody and glycan analyses) or used in pH determinations. Mycelia for cell dry weight determinations were rinsed once with DDIW and dried at +100.degree. C. for 20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIW and stored to -20.degree. C.
Generation of Pmt3 Deletion Strains M522 and M523
[0373] Generation of M317 is described in Example 1 above.
[0374] To remove vector sequence plasmid pTTv187 (.DELTA.pmt3-pyr4) was digested with PmeI+XbaI and the 4.1 kb fragment purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 .mu.g of the pmt3 deletion cassette was used to transform M317. Protoplast preparation and transformation were carried out essentially according to Penttila et al., 1987, Gene 61:155-164 and Gruber et al, 1990, Current Genetics 18:71-76 for pyr4 selection.
[0375] 200 colonies were picked as selective streaks. 59 transformants were screened by PCR using the primers in Table 20 for the correct integration of the deletion cassette using standard laboratory methods. Three putative deletion clones were purified to single cell clones. Purified clones were rescreened for correct integration and for deletion of pmt3 ORF (primers on Table 19). Two clones (several parallels) were pure deletants (i.e. no signal with ORF primers).
TABLE-US-00021 TABLE 20 Primers for screening integration of deletion cassette pTTv187 and for deletion of protein O- mannosyltransferase 3 (pmt3, TreID22527) from M317. Primer Sequence T292_22527_5int ACGGGAGATCTCGGAAAA (SEQ ID NO: 122) T026_Pyr4_orf_5rev2 CCATGAGCTTGAACAGGTAA (SEQ ID NO: 123) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 124) T293_22527_3int ATGAAGCTCAGCCTGTGG (SEQ ID NO: 125) T649_22527 orf 5' GGATCGGCCACTGCCACCAC probe (SEQ ID NO: 126) T650_22527 orf 3' GCCCACTTCTCTGCGCGTGT probe (SEQ ID NO: 127)
[0376] Deletion of pmt3 was verified by Southern analyses. DNA for Southern analyses was extracted with Easy-DNA kit for genomic DNA isolation (Invitrogen) essentially according to the manufacturer's instructions.
[0377] Southern analyses were essentially performed as described in Example 1. Fragments for probes were produced by PCR using the primers listed in Table 21 using a T. reesei wild type strain QM6a (ATCC13631) as the template for the ORF probe and plasmid pTTv187 for the 5' and 3' flank probes. PCR products were separated with agarose gel electrophoresis and correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using standard laboratory methods.
TABLE-US-00022 TABLE 21 Primers for production of probe fragments used in Southern analyses of protein O- mannosyltransferase 3 (pmt3, TreID22527) deletion strains. Primer Sequence T645_22527 5' flank TGGCAGATGCCGAAAGGCGG probe F (SEQ ID NO: 128) T646_22527 5' flank TGGCAACCAGCTGTGGCTCC probe R (SEQ ID NO: 129) T647_22527 3' flank CGGCCGCACTCGGCTTCTTT probe F (SEQ ID NO: 130) T648_22527 3' flank GAGTGGGCTAGGCGCAACGG probe R (SEQ ID NO: 131) T874_pmt3_orf_f3 CTCTGCGCGTGTTGTGG (SEQ ID NO: 132) T875_pmt3_orf_r3 TAAGGGTGCGGATTCGG (SEQ ID NO: 133)
[0378] Eight of the clones did not hybridise with pmt3 ORF probe (Data not shown) indicating successful deletion of pmt3. One clone (33-37K) hybridised with pmt3 ORF probe even though the signal size did not correspond to those from parental strains suggesting rearrangement in the pmt3 locus. Analyses using 5' and 3' flank probes revealed that the eight .DELTA.pmt3 clones were single integrants (Data not shown). One clone (33-37K) gave incorrect or additional signals suggesting rearrangements in the pmt3 locus and multiple integrations of the deletion cassette. Two pure clones have been stored for future use (M522; 33-34A and M523; 33-188A-a).
Analyses of .DELTA.Pmt3 Strains M522 and M523
[0379] 24-well plate cultivation of T. reesei strain M304 and eight pmt3 deletion strains (33-34S/M522, 33-34T, 33-34U, 33-34O, 33-188A-a/M523, 33-188B-a, 33-188C-a and 33-188D-a) was carried out in Trichoderma minimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, 9 g/l casamino acids, pH 5.5 at +28.degree. C., 800 rpm with humidity control. Samples were collected on days 3, 5 and 6 by centrifugation. Supernatant samples were stored to -20.degree. C. Mycelia for cell dry weight determinations were rinsed once with DDIW and dried at +100.degree. C. for 20-24 h. Mycelia for genomic DNA extraction were rinsed twice with DDIW and stored to -20.degree. C.
[0380] O-mannosylation level analysis was performed to pmt3 deletion strains as to flask cultures of pmt1 deletion strains. No difference was observed in O-mannosylation compared to parental strain M304.
Example 5
Pmt Homologs
[0381] T. reesei pmt homologs were identified from other organisms.
[0382] BLAST searches were conducted using the National Center for Biotechnology Information (NCBI) non-redundant amino acid database using the Trichoderma reesei PMT amino acid sequences as queries. Sequence hits from the BLAST searches were aligned using the ClustalW2 alignment tool provided by EBI. Phylogenetic trees were generated using average distance with BLOSUM62 after aligning the sequences in the Clustal Omega alignment tool.
[0383] A phylogenetic tree and a partial sequence alignment of the results of the PMT BLAST searches are depicted in FIGS. 5 and 6, respectively.
Sequence CWU
1
1
19712322DNATrichoderma reesei 1atggctcgaa gtccaacgcc gcagggcagc ctgcgacagc
ggaacgttgc gtccaagcag 60gcgcctgtcg agtcggcatt cgttcccgag gtcgagctcg
acaagctctc caaggccgct 120ctgtcgtcgc gccgaaacat ccagagaggc gagctcgagc
acaagcttgc cctgacgctg 180gtgacgatcc tcggctttgt cacgcgattc tggggcatca
gccaccccga cgaggtcgtc 240tttgacgagg tgcattttgg aaagttcgcc tcctactacc
tccagcgaac ctacttcttc 300gacgtccacc cccctttcgc caagctgctc ttcgccttcg
ttggctggct ggttggctac 360gacggtcact tccacttcga caacattggc gactcctacg
tggccaacaa ggtgccctat 420gtcgccttcc gagccttgcc cgccttcctc ggcgcattga
ctgtgtcggt cacatacctc 480atcatgtggg agtctggcta tagtgtgccg gcttgccttg
tcgcgaccgg cctgatcctc 540ctggacaatg cgcacattgg ccagacccgc ctcattctgc
tcgacgccac cctggtgctc 600gccatggcct gcagtctctt gttctacatc aagttctaca
agctgcggca cgagcccttt 660agccgcaagt ggtggaagtg gctcatcctg accggctttg
cgctgtcgtg cgacatctcg 720accaagtatg tcggtctctt tgcctttgtc accattggct
ccgccgtcat cattgatctg 780tgggatcttt tggatatcaa gcgccgctat ggagccatca
gcatgccaga gtttggaaag 840cactttgcag cccgcgcctt tggcctcatc atcttgccct
tcctcttcta cctcttctgg 900ttccaggtgc acttttccgt cctgacccga tccggtcccg
gcgacgactt catgactccc 960gagttccagg agacgttgag cgacaacgtc atgctggcaa
gcgccgtcga catccagtac 1020tacgatacca tcaccatcag gcacaaggag accaaggcgt
atcttcacag ccacaccgac 1080acctaccctc tgcgatatga cgacggccgc atctccagcc
aaggccaaca ggtcaccggc 1140tacccccaca acgacaccaa caactactgg cagatcctcc
ctgccgacaa tgaccagaag 1200ctcggccgta acgttaagaa tcaagacttg gtgcgacttc
gacacattgt cacggacaag 1260atcctgctct cccatgatgt cgcctcgccc tactacccta
ccaaccagga gttcacctgt 1320gtgacccccg aggaagcatt cggcgagcgc caaaacgaca
ctctgttcga gatccggatt 1380gagggaggca agaccggcca ggacttcaag accgttgcca
gccacttcaa gctcattcac 1440ttccccagca aggtggccat gtggactcat accacgcccc
ttcccgagtg ggcctacagg 1500cagcaggaaa tcaacggcaa caagcaaatc actcccagct
ccaacgtctg gattgccgaa 1560gacattcctt cgctcccgga agacgacgct cgccgccaca
aggagcagcg caaggtcaag 1620tcgctgccgt tcctccgcaa gtggtttgag ctgcagaggt
ccatgttcta ccacaacaac 1680aagctgacca gcagccaccc ctactccagc cagccctacc
actggccatt cctcctccgc 1740ggagtgagct tctggacgca gaatgacaca cgccagcaaa
tctactttgt gggcaacccc 1800atcggctggt ggcttgccag cagtctgctg gctgtgtttg
ccggcatcat tggagctgat 1860caggtctcgc tgcgccgagg catcgatgct ctggatcacc
gcacccgctc ccgactgtac 1920aactctaccg gcttcttctt ccttgcctgg gccacccact
acttcccctt tttcctcatg 1980ggtcgtcagc tgttcttgca tcactacttg cctgcccatt
tggcgtcctg cctggtcacg 2040ggctccctcg tcgagttcat ctttaacacg gacccggcag
acgaggagcc ttcgcgatcc 2100aaaaacccca aggctactgg tcctcggaga cacatcacgg
ctcgcgagcg gtttgctggc 2160aagagcatgg ccggtgcctg gatcgcttgc tttgtgattc
tcgctgccgc cgcggctagc 2220tggtacttct tcttgccgtt gacgtatggc taccccggac
tgtctgttga ggaggttctc 2280aggagaaagt ggcttggata tgatcttcac tttgccaagt
ag 23222773PRTTrichoderma reesei 2Met Ala Arg Ser
Pro Thr Pro Gln Gly Ser Leu Arg Gln Arg Asn Val 1 5
10 15 Ala Ser Lys Gln Ala Pro Val Glu Ser
Ala Phe Val Pro Glu Val Glu 20 25
30 Leu Asp Lys Leu Ser Lys Ala Ala Leu Ser Ser Arg Arg Asn
Ile Gln 35 40 45
Arg Gly Glu Leu Glu His Lys Leu Ala Leu Thr Leu Val Thr Ile Leu 50
55 60 Gly Phe Val Thr Arg
Phe Trp Gly Ile Ser His Pro Asp Glu Val Val 65 70
75 80 Phe Asp Glu Val His Phe Gly Lys Phe Ala
Ser Tyr Tyr Leu Gln Arg 85 90
95 Thr Tyr Phe Phe Asp Val His Pro Pro Phe Ala Lys Leu Leu Phe
Ala 100 105 110 Phe
Val Gly Trp Leu Val Gly Tyr Asp Gly His Phe His Phe Asp Asn 115
120 125 Ile Gly Asp Ser Tyr Val
Ala Asn Lys Val Pro Tyr Val Ala Phe Arg 130 135
140 Ala Leu Pro Ala Phe Leu Gly Ala Leu Thr Val
Ser Val Thr Tyr Leu 145 150 155
160 Ile Met Trp Glu Ser Gly Tyr Ser Val Pro Ala Cys Leu Val Ala Thr
165 170 175 Gly Leu
Ile Leu Leu Asp Asn Ala His Ile Gly Gln Thr Arg Leu Ile 180
185 190 Leu Leu Asp Ala Thr Leu Val
Leu Ala Met Ala Cys Ser Leu Leu Phe 195 200
205 Tyr Ile Lys Phe Tyr Lys Leu Arg His Glu Pro Phe
Ser Arg Lys Trp 210 215 220
Trp Lys Trp Leu Ile Leu Thr Gly Phe Ala Leu Ser Cys Asp Ile Ser 225
230 235 240 Thr Lys Tyr
Val Gly Leu Phe Ala Phe Val Thr Ile Gly Ser Ala Val 245
250 255 Ile Ile Asp Leu Trp Asp Leu Leu
Asp Ile Lys Arg Arg Tyr Gly Ala 260 265
270 Ile Ser Met Pro Glu Phe Gly Lys His Phe Ala Ala Arg
Ala Phe Gly 275 280 285
Leu Ile Ile Leu Pro Phe Leu Phe Tyr Leu Phe Trp Phe Gln Val His 290
295 300 Phe Ser Val Leu
Thr Arg Ser Gly Pro Gly Asp Asp Phe Met Thr Pro 305 310
315 320 Glu Phe Gln Glu Thr Leu Ser Asp Asn
Val Met Leu Ala Ser Ala Val 325 330
335 Asp Ile Gln Tyr Tyr Asp Thr Ile Thr Ile Arg His Lys Glu
Thr Lys 340 345 350
Ala Tyr Leu His Ser His Thr Asp Thr Tyr Pro Leu Arg Tyr Asp Asp
355 360 365 Gly Arg Ile Ser
Ser Gln Gly Gln Gln Val Thr Gly Tyr Pro His Asn 370
375 380 Asp Thr Asn Asn Tyr Trp Gln Ile
Leu Pro Ala Asp Asn Asp Gln Lys 385 390
395 400 Leu Gly Arg Asn Val Lys Asn Gln Asp Leu Val Arg
Leu Arg His Ile 405 410
415 Val Thr Asp Lys Ile Leu Leu Ser His Asp Val Ala Ser Pro Tyr Tyr
420 425 430 Pro Thr Asn
Gln Glu Phe Thr Cys Val Thr Pro Glu Glu Ala Phe Gly 435
440 445 Glu Arg Gln Asn Asp Thr Leu Phe
Glu Ile Arg Ile Glu Gly Gly Lys 450 455
460 Thr Gly Gln Asp Phe Lys Thr Val Ala Ser His Phe Lys
Leu Ile His 465 470 475
480 Phe Pro Ser Lys Val Ala Met Trp Thr His Thr Thr Pro Leu Pro Glu
485 490 495 Trp Ala Tyr Arg
Gln Gln Glu Ile Asn Gly Asn Lys Gln Ile Thr Pro 500
505 510 Ser Ser Asn Val Trp Ile Ala Glu Asp
Ile Pro Ser Leu Pro Glu Asp 515 520
525 Asp Ala Arg Arg His Lys Glu Gln Arg Lys Val Lys Ser Leu
Pro Phe 530 535 540
Leu Arg Lys Trp Phe Glu Leu Gln Arg Ser Met Phe Tyr His Asn Asn 545
550 555 560 Lys Leu Thr Ser Ser
His Pro Tyr Ser Ser Gln Pro Tyr His Trp Pro 565
570 575 Phe Leu Leu Arg Gly Val Ser Phe Trp Thr
Gln Asn Asp Thr Arg Gln 580 585
590 Gln Ile Tyr Phe Val Gly Asn Pro Ile Gly Trp Trp Leu Ala Ser
Ser 595 600 605 Leu
Leu Ala Val Phe Ala Gly Ile Ile Gly Ala Asp Gln Val Ser Leu 610
615 620 Arg Arg Gly Ile Asp Ala
Leu Asp His Arg Thr Arg Ser Arg Leu Tyr 625 630
635 640 Asn Ser Thr Gly Phe Phe Phe Leu Ala Trp Ala
Thr His Tyr Phe Pro 645 650
655 Phe Phe Leu Met Gly Arg Gln Leu Phe Leu His His Tyr Leu Pro Ala
660 665 670 His Leu
Ala Ser Cys Leu Val Thr Gly Ser Leu Val Glu Phe Ile Phe 675
680 685 Asn Thr Asp Pro Ala Asp Glu
Glu Pro Ser Arg Ser Lys Asn Pro Lys 690 695
700 Ala Thr Gly Pro Arg Arg His Ile Thr Ala Arg Glu
Arg Phe Ala Gly 705 710 715
720 Lys Ser Met Ala Gly Ala Trp Ile Ala Cys Phe Val Ile Leu Ala Ala
725 730 735 Ala Ala Ala
Ser Trp Tyr Phe Phe Leu Pro Leu Thr Tyr Gly Tyr Pro 740
745 750 Gly Leu Ser Val Glu Glu Val Leu
Arg Arg Lys Trp Leu Gly Tyr Asp 755 760
765 Leu His Phe Ala Lys 770
3944PRTTrichoderma reesei 3Met Ala Lys Ala Thr Ala Arg Gly Arg Ser Pro
Gln Pro Pro Leu Val 1 5 10
15 Ala Glu Lys Met Pro Val Ala Val Thr Ala Pro Val Ala Ser Ser Lys
20 25 30 Ser Lys
Ala Ala Lys Lys Asn Ser Ser Tyr Arg Ser Asp Gly Val Ala 35
40 45 Asp Asn Asp Val Phe Leu Leu
Pro Gly Ala Asp Tyr Val Ala Ala Leu 50 55
60 Gly Val Thr Val Leu Ala Thr Ile Val Arg Leu Phe
Lys Ile Tyr Thr 65 70 75
80 Pro Thr Ser Val Val Phe Asp Glu Val His Phe Gly Gly Phe Ala Ser
85 90 95 Lys Tyr Ile
Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala 100
105 110 Lys Met Leu Ile Ala Leu Thr Gly
Trp Leu Ala Gly Phe Asp Gly Asn 115 120
125 Phe Asp Phe Lys Asp Ile Gly Lys Asp Tyr Leu Glu Pro
Gly Val Pro 130 135 140
Tyr Val Ala Met Arg Met Phe Pro Ala Val Cys Gly Ile Leu Leu Ala 145
150 155 160 Pro Phe Met Phe
Phe Thr Leu Lys Ala Val Gly Cys Arg Thr Thr Thr 165
170 175 Ala Ile Leu Gly Ala Ser Phe Ile Ile
Phe Glu Asn Gly Leu Leu Thr 180 185
190 Gln Ala Arg Leu Ile Leu Leu Asp Ser Pro Leu Val Ala Ala
Thr Ala 195 200 205
Phe Thr Ala Met Ser Phe Asn Cys Phe Thr Asn Gln His Glu Gln Gly 210
215 220 Pro Asp Lys Ala Phe
Ser Leu Ser Trp Trp Phe Trp Leu Ala Met Thr 225 230
235 240 Gly Leu Gly Leu Gly Ile Thr Ser Ser Ile
Lys Trp Val Gly Leu Phe 245 250
255 Thr Ile Ala Trp Val Gly Ser Leu Thr Leu Val Gln Leu Trp Val
Leu 260 265 270 Leu
Gly Asp Ser Lys Asn Val Ser Met Arg Leu Trp Phe Lys His Phe 275
280 285 Met Ala Arg Val Phe Cys
Leu Ile Ile Ile Pro Leu Thr Phe Tyr Leu 290 295
300 Ser Met Phe Ala Ile His Phe Leu Cys Leu Thr
Asn Pro Gly Glu Gly 305 310 315
320 Asp Gly Phe Met Ser Ser Glu Phe Gln Ala Thr Leu Asn Ser Lys Gly
325 330 335 Met Lys
Asp Val Pro Ala Asp Val Val Phe Gly Ser Arg Val Thr Ile 340
345 350 Arg His Val Asn Thr Gln Gly
Gly Tyr Leu His Ser His Pro Leu Met 355 360
365 Tyr Pro Thr Gly Ser Leu Gln Gln Gln Ile Thr Leu
Tyr Pro His Lys 370 375 380
Asp Glu Asn Asn Ile Trp Ile Met Glu Asn Gln Thr Gln Pro Leu Gly 385
390 395 400 Val Asp Gly
Gln Pro Ile Asn Gly Thr Glu Ala Trp Asp Ala Leu Pro 405
410 415 Glu Val His His Val Val Asp Gly
Ser Val Ile Arg Leu Tyr His Lys 420 425
430 Pro Thr Phe Arg Arg Leu His Ser His Asp Val Arg Pro
Pro Val Thr 435 440 445
Glu Ala Glu Trp Gln Asn Glu Val Ser Ala Tyr Gly Tyr Glu Gly Phe 450
455 460 Glu Gly Asp Ala
Asn Asp Leu Phe Arg Val Glu Ile Val Lys Lys Gln 465 470
475 480 Ser Lys Gly Pro Leu Ala Lys Glu Arg
Leu Arg Thr Ile Glu Thr Lys 485 490
495 Phe Arg Leu Ile His Val Met Thr Gly Cys Ala Leu Phe Ser
His Lys 500 505 510
Val Lys Leu Pro Glu Trp Ala Ser Glu Gln Gln Glu Val Thr Cys Ala
515 520 525 Arg Gly Gly Ser
Leu Pro Asn Ser Ile Trp Tyr Ile Glu Tyr Asn Glu 530
535 540 His Pro Leu Leu Gly Asp Asp Val
Glu Lys Val Asn Tyr Ala Asn Pro 545 550
555 560 Gly Phe Phe Gly Lys Phe Trp Glu Leu His Lys Val
Met Trp Lys Thr 565 570
575 Asn Ala Gly Leu Thr Asp Ser His Ala Trp Asp Ser Arg Pro Pro Ser
580 585 590 Trp Pro Ile
Leu Arg Arg Gly Ile Asn Phe Trp Gly Lys His His Met 595
600 605 Gln Val Tyr Leu Leu Gly Asn Pro
Phe Ile Trp Trp Ser Ser Thr Ala 610 615
620 Ala Val Ala Ile Trp Val Ile Phe Lys Gly Val Ala Ile
Leu Arg Trp 625 630 635
640 Gln Arg Gly Cys Asn Asp Tyr Ala Ser Ser Thr Phe Lys Arg Phe Asp
645 650 655 Tyr Glu Ile Gly
Thr Ser Val Leu Gly Trp Ala Leu His Tyr Phe Pro 660
665 670 Phe Tyr Leu Met Glu Arg Gln Leu Phe
Leu His His Tyr Phe Pro Ala 675 680
685 Leu Tyr Phe Ala Ile Leu Ala Leu Cys Gln Met Phe Asp Phe
Ala Thr 690 695 700
Val Arg Ile Pro Ala Ala Leu Gly Tyr Arg Ser Thr Leu Ile Asn Arg 705
710 715 720 Val Gly Thr Val Ser
Leu Leu Val Ile Ser Ala Ala Val Phe Thr Leu 725
730 735 Phe Ala Pro Leu Ala Tyr Gly Thr Pro Trp
Thr Lys Ala Glu Cys Asn 740 745
750 Arg Val Lys Leu Phe Asp Lys Trp Asp Phe Asp Cys Asn Thr Phe
Leu 755 760 765 Asp
Asp Tyr Lys Ser Tyr Thr Leu Thr Ser Leu Ala Pro Ser Ser Ile 770
775 780 Ala Pro Ser Pro Pro Ala
Ala Asn Val Pro Val Val Asn Gln Glu Gln 785 790
795 800 Lys Pro Leu Ala Lys Gln Pro Glu Pro Val Ile
Ser Gln Ala Ala Val 805 810
815 Pro Gln Glu Pro Gln Ile Leu Ser Lys Glu Glu Lys Ile Glu Tyr Arg
820 825 830 Asp Gln
Asp Gly Asn Leu Leu Asn Asp Glu Gln Val Lys Ala Leu Gln 835
840 845 Gly Lys Val Glu Phe Lys Thr
Lys Tyr Glu Thr Lys Thr Arg Val Val 850 855
860 Asp Ala Gln Gly His Glu Ile Pro Val Pro Glu Gly
Gly Trp Pro Asp 865 870 875
880 Asp Met Ile Ala Gly Val Ala Pro Pro His Pro Asp Val Glu Gly Val
885 890 895 Asp Lys Glu
Thr Pro Lys Val Glu Ser Ala Glu Val Pro Lys Glu Ala 900
905 910 Ala Ala Ser Arg Asp Gly Glu Val
Glu Ala Glu Asn Leu Lys Ala Lys 915 920
925 Pro Ala Ser Glu Gly Gln Glu Val Glu Ala Thr Val Gln
Glu Glu Leu 930 935 940
4740PRTTrichoderma reesei 4Met Ala Ala Asp Lys Ala Ala Leu Ala Ser Gly
Ala Asp Leu Gly Asp 1 5 10
15 Gly Leu Arg Lys Arg Gln Ala Ala Ser Gln Ala Val Pro Ser Phe Ile
20 25 30 Pro Ala
Gln Thr Glu Asp Thr Lys Lys Leu Ala Lys Lys Asp Lys Thr 35
40 45 Phe Val Gln Val Leu Ala Asp
Trp Glu Ser Val Leu Ala Pro Leu Ile 50 55
60 Phe Thr Ala Val Ala Ile Phe Thr Arg Leu Tyr Lys
Ile Gly Leu Ser 65 70 75
80 Asn Ile Val Thr Trp Asp Glu Ala His Phe Gly Lys Phe Gly Ser Tyr
85 90 95 Tyr Ile Lys
His Glu Tyr Tyr Phe Asp Val His Pro Pro Leu Gly Lys 100
105 110 Met Leu Val Gly Leu Ser Gly Val
Leu Ala Gly Tyr Asn Gly Ser Phe 115 120
125 Glu Phe Lys Ser Gly Glu Gln Tyr Pro Glu Asp Val Asn
Tyr Thr Phe 130 135 140
Met Arg Ala Phe Asn Ala Ala Phe Gly Ile Ala Cys Ile Pro Met Ala 145
150 155 160 Tyr Phe Thr Ala
Lys Glu Leu Lys Leu Thr Arg Pro Ala Val Trp Phe 165
170 175 Val Thr Leu Met Val Leu Cys Glu Asn
Ser Tyr Thr Thr Ile Ser Arg 180 185
190 Phe Ile Leu Leu Asp Ser Met Leu Leu Cys Gly Thr Phe Ala
Thr Thr 195 200 205
Leu Cys Trp Ala Lys Phe His Asn Gln Arg His Asn Ser Phe Glu Pro 210
215 220 Glu Trp Phe Phe Trp
Leu Phe Met Thr Gly Leu Ser Ile Gly Cys Val 225 230
235 240 Cys Ser Val Lys Leu Val Gly Leu Phe Val
Thr Ala Leu Val Gly Leu 245 250
255 Tyr Thr Ile Glu Asp Leu Trp Arg Lys Tyr Gly Asp Arg Lys Met
Pro 260 265 270 Ile
Pro Val Leu Ala Ala His Phe Ser Ala Arg Val Val Gly Leu Ile 275
280 285 Ile Val Pro Phe Leu Ile
Tyr Met Leu Ser Phe Ala Leu His Phe Ala 290 295
300 Ile Leu Asp His Ser Gly Pro Gly Asp Ala Gln
Met Ser Ser Leu Phe 305 310 315
320 Gln Ala Asn Leu Lys Gly Thr Glu Val Gly Lys Asn Ser Pro Leu Glu
325 330 335 Ile Ala
Leu Gly Ser Arg Ala Thr Ile Lys Asn Met Gly Tyr Gly Gly 340
345 350 Gly Leu Leu His Ser His Val
Gln Thr Tyr Pro Glu Gly Ser Gly Gln 355 360
365 Gln Gln Val Thr Cys Tyr His His Lys Asp Ala Asn
Asn Asp Trp Phe 370 375 380
Phe Tyr Pro Asn Arg His Glu Pro Asp Tyr Asp Pro Glu Gly Glu Leu 385
390 395 400 Arg Phe Ile
Gly Asp Gly Ser Val Ile Arg Leu Ile His Ala Gln Thr 405
410 415 Gly Arg Asn Leu His Ser His Asp
Ile Asp Ala Pro Ile Thr Lys Ser 420 425
430 His Arg Glu Val Ser Ser Tyr Gly Asn Leu Thr Val Gly
Asp Glu Lys 435 440 445
Asp His Trp Lys Ile Glu Val Val Arg Asp Ala Ala Ser Arg Asp Arg 450
455 460 Ser Arg Ile Arg
Thr Leu Thr Thr Ala Phe Arg Leu Lys His Thr Val 465 470
475 480 Leu Gly Cys Tyr Leu Arg Ala Gly Asn
Val Asn Leu Pro Gln Trp Gly 485 490
495 Phe Lys Gln Ile Glu Val Thr Cys Asp Lys Gln Asn Asn Pro
Arg Asp 500 505 510
Thr Tyr Thr His Trp Asn Val Glu Ala His Trp Asn Asp Arg Leu Pro
515 520 525 Pro Ser Asp Pro
Gly Val Tyr Lys Ser Pro Phe Ile His Asp Phe Ile 530
535 540 His Leu Asn Val Ala Met Met Thr
Ser Asn Asn Ala Leu Val Pro Asp 545 550
555 560 Pro Asp Lys Gln Asp Asp Leu Ala Ser Gln Trp Trp
Gln Trp Pro Ile 565 570
575 Leu His Val Gly Leu Arg Met Cys Ser Trp Asp Asp Asn Ile Val Lys
580 585 590 Tyr Phe Leu
Leu Gly Asn Pro Phe Val Tyr Trp Ala Ser Thr Ala Ser 595
600 605 Leu Gly Ala Val Ala Leu Val Ile
Ala Trp Tyr Val Val Arg Trp Gln 610 615
620 Arg Gly Phe Lys Glu Leu Ser Asn Ser Glu Val Asp Gln
Ile His Tyr 625 630 635
640 Ala Gly Ile Tyr Pro Val Ile Gly Trp Phe Leu His Tyr Leu Pro Phe
645 650 655 Val Ile Met Ala
Arg Val Thr Tyr Val His His Tyr Tyr Pro Ala Leu 660
665 670 Tyr Phe Ala Ile Leu Ser Leu Gly Phe
Leu Val Asp Trp Val Leu Arg 675 680
685 Asn Arg Ala Ala Val Val Gln Gly Val Ala Tyr Gly Ile Leu
Tyr Thr 690 695 700
Val Val Ile Gly Leu Tyr Ile Leu Phe Met Pro Ile Cys Trp Gly Met 705
710 715 720 Thr Gly Ser Ser Lys
Gln Tyr Ser Tyr Leu Lys Trp Phe Asp Asn Trp 725
730 735 Arg Ile Ser Asp 740
5775PRTAspergillus oryzae 5Met Ser Gln Ser Pro Ser Pro Ser Leu Arg Lys
Arg Gly Gly Lys Lys 1 5 10
15 Glu Ala Ser Pro Gly Pro Ser Glu Val Ser Ser Pro Tyr Pro Thr Asn
20 25 30 Gln Gly
Ala Thr Pro Lys Pro Gln Ser Glu Trp Asp Tyr Arg Leu Ala 35
40 45 Ile Thr Val Leu Thr Val Leu
Ala Phe Ile Thr Arg Phe Tyr Arg Ile 50 55
60 Ser Tyr Pro Asp Glu Val Val Phe Asp Glu Val His
Phe Gly Lys Phe 65 70 75
80 Ala Ser Tyr Tyr Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro
85 90 95 Phe Gly Lys
Leu Leu Phe Ala Ala Val Gly Trp Leu Ile Gly Tyr Asp 100
105 110 Gly His Phe Leu Phe Glu Asn Ile
Gly Asp Ser Tyr Ile Asp Asn Lys 115 120
125 Val Pro Tyr Val Ala Phe Arg Ala Leu Pro Ala Thr Leu
Gly Ala Leu 130 135 140
Thr Val Pro Val Val Phe Leu Ile Met Trp Glu Ser Gly Tyr Ser Leu 145
150 155 160 Pro Ala Cys Val
Leu Ala Ala Gly Leu Val Leu Phe Asp Asn Ala His 165
170 175 Ile Gly Glu Asp Arg Leu Ile Leu Leu
Asp Ala Thr Leu Val Ile Thr 180 185
190 Met Ala Leu Ser Ile Leu Cys Tyr Val Arg Phe Tyr Lys Leu
Arg His 195 200 205
Glu Pro Phe Gly Arg Lys Trp Trp Lys Trp Leu Leu Leu Thr Gly Val 210
215 220 Ser Leu Ser Cys Val
Ile Ser Thr Lys Tyr Val Gly Val Phe Thr Phe 225 230
235 240 Val Thr Ile Gly Ala Ala Val Met Val Asp
Leu Trp Asn Leu Leu Asp 245 250
255 Ile Arg Arg Pro Ala Gly Ala Leu Ser Met Met Glu Trp Thr Lys
His 260 265 270 Phe
Ala Ala Arg Gly Phe Ala Leu Ile Val Val Pro Phe Phe Phe Tyr 275
280 285 Leu Phe Trp Phe Gln Val
His Phe Ala Ile Leu Thr Arg Ser Gly Pro 290 295
300 Gly Asp Asp Phe Met Thr Pro Glu Phe Gln Glu
Thr Leu Ser Asp Asn 305 310 315
320 Ala Leu Ala Ala Glu Ser Ile Gly Ile Gln Tyr Tyr Asp Ala Ile Thr
325 330 335 Ile Arg
His Lys Asp Thr Lys Val Phe Leu His Ser His Trp Glu Arg 340
345 350 Tyr Pro Leu Arg Tyr Asp Asp
Gly Arg Ile Ser Ser Gln Gly Gln Gln 355 360
365 Val Thr Gly Tyr Pro Phe Asn Asp Thr Asn Asn Gln
Trp Gln Ile Leu 370 375 380
Pro Thr Val Pro Leu Glu Asp Asn Glu Gly Gln Gly His Ser Val Lys 385
390 395 400 Asn Gly Asp
Leu Val Gln Leu Leu His Leu Gly Thr Asp Ser Ile Leu 405
410 415 Leu Thr His Asp Val Ala Ser Pro
Phe Tyr Pro Thr Asn Gln Glu Phe 420 425
430 Thr Thr Val Thr Lys Asp Val Ala Ser Gly Glu Arg His
Asn Glu Thr 435 440 445
Leu Phe Glu Ile Lys Ile Glu Asn Gly Lys Ala Gly Gln Glu Phe Arg 450
455 460 Thr Leu Ser Ser
His Phe Lys Leu Ile His Tyr Pro Thr Arg Val Ala 465 470
475 480 Met Trp Thr His Thr Thr Pro Leu Pro
Glu Trp Gly Phe Lys Gln Ala 485 490
495 Glu Ile Asn Gly Asn Lys Asn Val Leu Gln Thr Ser Asn Leu
Trp Tyr 500 505 510
Ala Glu Ser Ile Glu Ser Leu Glu Glu Asp Ser Pro Arg Lys Gln Lys
515 520 525 Glu Glu Arg Lys
Val Lys Gln Leu Pro Phe Leu Arg Lys Tyr Leu Glu 530
535 540 Leu Gln Arg Ala Met Phe Phe His
Asn Asn Ala Leu Thr Ser Ser His 545 550
555 560 Pro Tyr Ala Ser Glu Pro Phe Gln Trp Pro Phe Leu
Leu Arg Gly Val 565 570
575 Ser Phe Trp Thr Lys Asn Asp Thr Arg Glu Gln Ile Tyr Phe Leu Gly
580 585 590 Asn Pro Ile
Gly Trp Trp Ile Ala Ser Ser Leu Leu Ala Val Phe Ala 595
600 605 Gly Val Ile Gly Ala Asp Gln Leu
Ser Leu Arg Arg Gly Val Asp Ala 610 615
620 Val Glu Glu Ile Trp Gly Pro Gly Ala Arg Ser Arg Leu
Tyr Asn Ser 625 630 635
640 Thr Gly Phe Leu Phe Leu Cys Trp Gly Ala His Tyr Phe Pro Phe Trp
645 650 655 Leu Met Gly Arg
Gln Arg Phe Leu His His Tyr Leu Pro Ala His Leu 660
665 670 Ala Ser Cys Leu Val Thr Gly Ala Leu
Ile Glu Phe Ile Phe Asn Leu 675 680
685 Gln Pro Val Gln Ala Val Ile Asp Ser Glu Val Asp Pro Ser
Gly Lys 690 695 700
Ser Lys Ser Ile Arg Pro Arg His Phe Val Thr Ala Lys Glu Arg Met 705
710 715 720 Ser Arg Lys Ser Leu
Val Ala Cys Trp Ile Ala Thr Leu Ser Ile Leu 725
730 735 Ala Val Thr Val Trp Gly Phe Trp Phe Tyr
Ala Pro Leu Thr Tyr Gly 740 745
750 Thr Pro Gly Leu Asp Val Ala Gly Val Asn Ala Arg Arg Trp Leu
Gly 755 760 765 Tyr
Asp Leu His Phe Ala Lys 770 775 6775PRTAspergillus
niger 6Met Ser Ser Ser Pro Ser Pro Ser Leu Arg Lys Arg Gly Gly Lys Lys 1
5 10 15 Glu Ser Thr
Pro Val Pro Ala Asp Asn Phe Ser Ser Pro Leu Ser Lys 20
25 30 Ala Ser Ala Pro Arg Ser Glu Trp
Asp Tyr Trp Leu Ala Ile Ser Ile 35 40
45 Leu Thr Val Leu Ala Phe Val Thr Arg Phe Tyr Lys Ile
Ser Tyr Pro 50 55 60
Asn Glu Val Val Phe Asp Glu Val His Phe Gly Lys Phe Ala Ser Tyr 65
70 75 80 Tyr Leu Gln Arg
Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys 85
90 95 Leu Leu Phe Ala Phe Met Gly Trp Leu
Val Gly Tyr Asp Gly His Phe 100 105
110 Leu Phe Asp Asn Ile Gly Asp Ser Tyr Ile Glu His Gln Val
Pro Tyr 115 120 125
Val Ala Leu Arg Ala Met Pro Ala Thr Leu Gly Ala Leu Thr Val Pro 130
135 140 Val Val Phe Leu Ile
Met Trp Glu Ser Gly Tyr Ser Leu Pro Ala Cys 145 150
155 160 Val Leu Ser Ala Gly Leu Val Leu Phe Asp
Asn Ala His Ile Gly Glu 165 170
175 Asp Arg Leu Ile Leu Leu Asp Ala Ser Leu Val Leu Thr Met Ala
Leu 180 185 190 Ser
Ile Leu Cys Tyr Ile Arg Phe Tyr Lys Leu Arg His Glu Ala Phe 195
200 205 Gly Arg Lys Trp Trp Lys
Trp Leu Leu Leu Thr Gly Val Ser Leu Ser 210 215
220 Cys Val Ile Ser Thr Lys Tyr Val Gly Val Phe
Thr Phe Val Thr Ile 225 230 235
240 Gly Ser Ala Val Met Val Asp Leu Trp Asn Leu Leu Asp Ile Arg Arg
245 250 255 Arg Gly
Gly Ala Leu Thr Met Phe Gln Trp Gly Gln His Phe Val Ala 260
265 270 Arg Ala Phe Ala Leu Ile Ile
Val Pro Phe Phe Phe Tyr Leu Phe Trp 275 280
285 Phe Gln Val His Phe Ala Ile Leu Thr Arg Ser Gly
Pro Gly Asp Asp 290 295 300
Phe Met Thr Pro Glu Phe Gln Glu Thr Leu Ser Asp Asn Val Leu Ser 305
310 315 320 Ala Gln Ser
Ile Gly Ile Glu Tyr Tyr Asp Thr Ile Thr Met Lys His 325
330 335 Lys Asp Thr Lys Val Tyr Leu His
Ser His Leu Glu Arg Tyr Pro Leu 340 345
350 Arg Tyr Asp Asp Gly Arg Ile Ser Ser Gln Gly Gln Gln
Val Thr Gly 355 360 365
Tyr Pro Tyr Asn Asp Thr Asn Asn Gln Trp Gln Ile Ile Pro Thr Val 370
375 380 Pro Leu Asp Val
Thr Asp Thr Ser Gly His Lys Val Arg Asn Gly Asp 385 390
395 400 Val Val Gln Leu Arg His Met Gly Thr
Asp Thr Ile Leu Leu Thr His 405 410
415 Asp Val Ala Ser Pro Tyr Tyr Pro Thr Asn Gln Glu Phe Thr
Thr Val 420 425 430
Ser His Glu Val Ala Asn Gly Asp Arg His Asn Asp Thr Leu Phe Glu
435 440 445 Ile Lys Ile Glu
Asn Gly Lys Pro His Gln Glu Phe Arg Thr Leu Ser 450
455 460 Ser His Phe Lys Leu Ile His Met
Pro Thr Arg Val Ala Met Trp Thr 465 470
475 480 His Thr Thr Pro Leu Pro Asp Trp Ala Phe Lys Gln
Ala Glu Ile Asn 485 490
495 Gly Asn Lys Asn Ile Leu Gln Thr Ser Asn Leu Trp Phe Val Glu Ser
500 505 510 Ile Glu Ser
Leu Glu Glu Asp Ser Pro Arg Leu Val Lys Glu Glu Arg 515
520 525 Gln Val Lys His Leu Pro Phe Phe
Arg Lys Tyr Leu Glu Leu Gln Arg 530 535
540 Ala Met Phe Phe His Asn Asn Ala Leu Thr Ser Ser His
Pro Tyr Ala 545 550 555
560 Ser Glu Pro Phe Gln Trp Pro Phe Leu Leu Arg Gly Val Ser Phe Trp
565 570 575 Thr Lys Asn Asp
Thr Arg Glu Gln Ile Tyr Phe Leu Gly Asn Pro Val 580
585 590 Gly Trp Trp Ile Ala Ser Ser Leu Leu
Ala Val Phe Ala Gly Val Ile 595 600
605 Gly Ala Asp Gln Leu Ser Leu Arg Arg Gly Val Asp Ala Val
Glu Glu 610 615 620
Ile Trp Gly Gln Gly Ser Arg Ser Arg Leu Tyr Asn Ser Met Gly Phe 625
630 635 640 Leu Phe Leu Cys Trp
Ala Ala His Tyr Phe Pro Phe Trp Leu Met Gly 645
650 655 Arg Gln Arg Phe Leu His His Tyr Leu Pro
Ala His Leu Ala Ser Ala 660 665
670 Leu Val Ala Gly Ala Leu Ile Glu Phe Ile Phe Asn Leu Glu Pro
Leu 675 680 685 Ser
Val Ile Gln Arg Val Arg Ser Glu Asp Asp Pro Ser Gly Lys Ala 690
695 700 Lys Ala Ser Ala Ser Val
Gly Arg Phe Val Thr Ala Lys Glu Arg Met 705 710
715 720 Gly Thr Lys Ser Leu Leu Ala Gly Trp Ile Ala
Thr Leu Val Ile Leu 725 730
735 Ala Gly Thr Ile Tyr Gly Phe Val Phe Tyr Ala Pro Leu Thr Tyr Gly
740 745 750 Thr Pro
Gly Leu Asp Val Pro Gly Ile Leu Ala Arg Lys Trp Leu Gly 755
760 765 Tyr Asp Leu His Phe Ala Lys
770 775 7773PRTAspergillus nidulans 7Met Ser Ser Ser
Pro Ser Leu Arg Lys Arg Gly Gly Lys Arg Glu Asp 1 5
10 15 Thr Pro Val Pro Ser Asp Arg Ser Phe
Ala Pro Ser Ala Ser Gln Leu 20 25
30 Gly Ala Ala Ser Arg Ser Ser Glu Trp Asp Tyr Arg Leu Ala
Ile Thr 35 40 45
Ile Leu Thr Val Leu Ala Phe Ile Thr Arg Phe Tyr Lys Ile Ser Tyr 50
55 60 Pro Asp Gln Val Val
Phe Asp Glu Val His Phe Gly Lys Phe Ala Ser 65 70
75 80 Tyr Tyr Leu Arg Arg Thr Tyr Phe Phe Asp
Val His Pro Pro Phe Ala 85 90
95 Lys Leu Leu Leu Ala Phe Thr Gly Trp Leu Val Gly Tyr Asp Gly
His 100 105 110 Phe
Leu Phe Glu Asn Ile Gly Asp Ser Tyr Ile Asp Asn Lys Val Pro 115
120 125 Tyr Val Ala Leu Arg Ala
Met Pro Ala Val Leu Gly Ala Leu Thr Ile 130 135
140 Pro Val Val Phe Leu Ile Met Trp Glu Ser Gly
Tyr Ser Leu Pro Ala 145 150 155
160 Cys Val Leu Ala Ser Gly Leu Val Leu Phe Asp Asn Ala His Val Gly
165 170 175 Glu Asp
Arg Leu Ile Leu Leu Asp Ser Thr Leu Val Ile Thr Met Ala 180
185 190 Leu Ser Ile Leu Cys Tyr Ile
Arg Phe Tyr Lys Leu Arg His Glu Pro 195 200
205 Phe Gly Arg Lys Trp Trp Lys Trp Leu Leu Leu Thr
Gly Val Ser Leu 210 215 220
Ser Cys Val Ile Ser Thr Lys Tyr Val Gly Val Phe Thr Phe Val Thr 225
230 235 240 Ile Gly Ser
Ala Val Met Val Asp Leu Trp Asn Leu Leu Asp Ile Arg 245
250 255 Arg Gln Gly Gly Ala Leu Thr Met
Phe Glu Trp Thr Lys His Phe Ala 260 265
270 Ala Arg Phe Phe Ser Leu Ile Val Val Pro Phe Phe Phe
Tyr Leu Phe 275 280 285
Trp Phe Gln Val His Phe Ala Ile Leu Thr His Ser Gly Pro Gly Asp 290
295 300 Asp Phe Met Thr
Pro Ala Phe Gln Glu Thr Leu Ser Asp Asn Ala Met 305 310
315 320 Ala Ala Gln Ser Val Ser Ile Glu Tyr
Phe Asp Thr Ile Thr Met Arg 325 330
335 His Lys Asp Thr Lys Val Phe Leu His Ser His Ser Asp Thr
Tyr Pro 340 345 350
Leu Arg Tyr Asp Asp Gly Arg Ile Ser Ser Gln Gly Gln Gln Val Thr
355 360 365 Gly Tyr Pro Tyr
Asn Asp Thr Asn Asn His Trp Gln Ile Ile Pro Thr 370
375 380 Val Pro Leu Asp Glu Thr Asp Glu
Lys Ser Arg Lys Val Arg Asn Gly 385 390
395 400 Asp Ile Val Gln Leu Arg His Val Ala Thr Asp Thr
Ile Leu Leu Thr 405 410
415 His Asp Val Ala Ser Pro Tyr Tyr Pro Thr Asn Gln Glu Phe Thr Thr
420 425 430 Val Ser His
Glu Leu Ala Asp Gly Lys Arg His Asn Asp Thr Leu Phe 435
440 445 Glu Ile Arg Val Glu His Gly Lys
Ser Lys Gln Glu Phe Arg Thr Leu 450 455
460 Ser Ser Gln Phe Lys Leu Val His Val Pro Thr Lys Val
Ala Met Trp 465 470 475
480 Thr His Thr Thr Pro Leu Pro Asp Trp Ala Tyr Lys Gln Ala Glu Ile
485 490 495 Asn Gly Asn Lys
Asn Val Leu Gln Ser Ser Asn Ile Trp Tyr Val Glu 500
505 510 Ala Ile Glu Ser Leu Glu Glu Asp Ser
Pro Arg Leu Lys Lys Glu Glu 515 520
525 Arg Lys Val Lys His Leu Pro Phe Trp Arg Lys Tyr Ile Glu
Leu Gln 530 535 540
Arg Ala Met Phe Phe His Asn Asn Ala Leu Thr Ser Ser His Pro Tyr 545
550 555 560 Ala Ser Glu Pro Phe
Gln Trp Pro Phe Leu Leu Arg Gly Val Ser Phe 565
570 575 Trp Thr Lys Ser Asp Thr Arg Glu Gln Ile
Tyr Phe Leu Gly Asn Pro 580 585
590 Val Gly Trp Trp Ile Ser Ser Ser Leu Leu Ala Val Phe Ala Gly
Val 595 600 605 Ile
Gly Ala Asp Gln Leu Ser Leu Arg Arg Gly Val Asp Ala Val Glu 610
615 620 Glu Ile Trp Gly Pro Gly
Ser Arg Ser Arg Leu Tyr Asn Ser Thr Gly 625 630
635 640 Phe Leu Phe Leu Cys Trp Ala Ala His Tyr Phe
Pro Phe Trp Leu Met 645 650
655 Gly Arg Gln Arg Phe Leu His His Tyr Leu Pro Ala His Val Ala Ser
660 665 670 Ala Leu
Val Thr Gly Ala Leu Ile Glu Phe Ile Phe Asn Ile Gln Pro 675
680 685 Ile Ser Val Pro Ala Thr Ile
Pro Val Ala Ala Asp Asp Pro Thr Gly 690 695
700 Lys Gly Lys Thr Arg Arg Phe Val Thr Ala Arg Glu
Arg Met Gly Val 705 710 715
720 Lys Ser Ile Val Ala Gly Trp Ile Ala Ser Leu Thr Ile Leu Ala Ala
725 730 735 Thr Ile Trp
Gly Phe Trp Phe Phe Ala Pro Leu Thr Tyr Gly Thr Pro 740
745 750 Gly Leu Asp Val Ala Gln Val Asn
Ala Arg Lys Trp Leu Gly Tyr Asp 755 760
765 Leu His Phe Ala Lys 770
8774PRTTrichoderma virens 8Met Ala Arg Thr Pro Thr Pro Gln Pro Pro Ser
Leu Arg Gln Arg Asn 1 5 10
15 Val Ala Ser Lys Gln Pro Val Ser Glu Ala Thr Phe Ala Pro Glu Val
20 25 30 Glu Leu
Asp Lys Leu Ser Lys Ala Ala Ala Ser Ser Arg Gln Asn Ile 35
40 45 Gln Arg Gly Glu Thr Glu His
Arg Val Ala Leu Thr Leu Val Thr Ile 50 55
60 Leu Gly Phe Val Thr Arg Phe Trp Gly Ile Ser His
Pro Asp Glu Val 65 70 75
80 Val Phe Asp Glu Val His Phe Gly Lys Phe Ala Ser Tyr Tyr Leu Gln
85 90 95 Arg Thr Tyr
Phe Phe Asp Val His Pro Pro Phe Ala Lys Leu Leu Phe 100
105 110 Ala Phe Val Gly Trp Leu Val Gly
Tyr Asp Gly His Phe His Phe Glu 115 120
125 Asn Ile Gly Asp Ser Tyr Ile Ala Asn Lys Val Pro Tyr
Val Ala Phe 130 135 140
Arg Ala Leu Pro Ala Phe Leu Gly Ala Leu Thr Val Ser Val Thr Tyr 145
150 155 160 Leu Ile Met Trp
Glu Ser Gly Tyr Ser Val Pro Ala Cys Leu Val Ala 165
170 175 Thr Gly Leu Ile Leu Leu Asp Asn Ala
His Ile Gly Gln Thr Arg Leu 180 185
190 Ile Leu Leu Asp Ala Thr Leu Val Leu Ala Met Ala Cys Ser
Leu Leu 195 200 205
Phe Tyr Ile Lys Phe Tyr Lys Leu Arg His Glu Pro Phe Ser Arg Lys 210
215 220 Trp Trp Lys Trp Leu
Val Leu Thr Gly Phe Ala Leu Ser Cys Asp Ile 225 230
235 240 Ser Thr Lys Tyr Val Gly Leu Phe Ala Phe
Val Thr Ile Gly Ser Ala 245 250
255 Val Ile Ile Asp Leu Trp Glu Leu Leu Asp Ile Arg Arg Pro Gly
Gly 260 265 270 Ala
Ile Ser Leu Pro Leu Phe Gly Lys His Phe Ala Ala Arg Ala Val 275
280 285 Gly Leu Ile Ile Leu Pro
Phe Leu Phe Tyr Leu Phe Trp Phe Gln Val 290 295
300 His Phe Ala Val Leu Thr Arg Ser Gly Pro Gly
Asp Asp Phe Met Ser 305 310 315
320 Pro Glu Phe Gln Glu Thr Leu Ser Asp Asn Val Met Leu Ala Ser Ala
325 330 335 Val Asp
Ile Gln Tyr Tyr Asp Thr Ile Thr Ile Arg His Lys Glu Thr 340
345 350 Lys Ala Tyr Leu His Ser His
Leu Asp Thr Tyr Pro Leu Arg Tyr Asp 355 360
365 Asp Gly Arg Ile Ser Ser Gln Gly Gln Gln Val Thr
Gly Tyr Pro His 370 375 380
Asn Asp Thr Asn Asn Tyr Trp Gln Ile Ile Pro Ala Ser Asn Asp Gln 385
390 395 400 Lys Leu Gly
Arg Ile Val Arg Asn Gln Glu Leu Val Arg Leu Arg His 405
410 415 Ile Val Thr Asp Lys Ile Leu Leu
Ser His Asp Val Ala Ser Pro Tyr 420 425
430 Tyr Pro Thr Asn Gln Glu Phe Thr Ala Val Ser Ala Glu
Glu Ala Tyr 435 440 445
Gly Asp Arg Leu Asn Asp Thr Leu Phe Glu Ile Arg Ile Glu Gly Gly 450
455 460 Lys Pro Asn Gln
Asp Phe Lys Thr Ile Ala Ser His Phe Lys Leu Ile 465 470
475 480 His Phe Pro Ser Lys Val Ala Met Trp
Thr His Thr Thr Pro Leu Pro 485 490
495 Glu Trp Ala Tyr Arg Gln Gln Glu Ile Asn Gly Asn Lys Gln
Ile Thr 500 505 510
Pro Ser Ser Asn Val Trp Ile Ala Glu Asp Ile Pro Ser Leu Pro Glu
515 520 525 Asp His Ser Arg
Arg Gln Lys Glu Glu Arg Lys Val Lys Ser Leu Pro 530
535 540 Phe Leu Arg Lys Trp Phe Glu Leu
Gln Arg Ser Met Phe Tyr His Asn 545 550
555 560 Asn Lys Leu Thr Ser Ser His Pro Tyr Ser Ser Gln
Pro Tyr His Trp 565 570
575 Pro Phe Leu Leu Arg Gly Val Ser Phe Trp Thr Gln Asn Asp Thr Arg
580 585 590 Gln Gln Ile
Tyr Phe Val Gly Asn Pro Ile Gly Trp Trp Leu Ala Ser 595
600 605 Gly Leu Leu Ala Val Phe Ala Gly
Ile Ile Gly Ala Asp Gln Val Ser 610 615
620 Leu Arg Arg Gly Ile Asp Ala Leu Asp His Arg Thr Arg
Ser Arg Leu 625 630 635
640 Tyr Asn Ser Thr Gly Phe Phe Trp Leu Ala Trp Ala Thr His Tyr Phe
645 650 655 Pro Phe Phe Leu
Met Gly Arg Gln Leu Phe Leu His His Tyr Leu Pro 660
665 670 Ala His Leu Ala Ser Cys Leu Val Thr
Gly Ser Leu Val Glu Phe Ile 675 680
685 Phe Asn Thr Asp Pro Ala Asp Glu Glu Pro Ser Arg Ala Thr
Asn Pro 690 695 700
Arg Ala Ser Gly Pro Lys Arg His Ile Thr Ala Arg Glu Arg Phe Ala 705
710 715 720 Gly Lys Ser Met Ala
Gly Ala Trp Ile Ala Cys Phe Val Ile Leu Thr 725
730 735 Val Ala Ala Ala Ser Trp Tyr Phe Phe Leu
Pro Leu Thr Tyr Gly Tyr 740 745
750 Pro Gly Leu Ser Val Asp Glu Val Asn Arg Arg Lys Trp Leu Gly
Tyr 755 760 765 Asp
Leu His Phe Ala Lys 770 9771PRTTrichoderma atroviride
9Met Ala Arg Ala Ser Thr Pro Gln Gly Ser Leu Arg Gln Arg Gly Val 1
5 10 15 Ala Ser Lys Gln
Thr Leu Ser Glu Ser Thr Phe Ala Pro Glu Val Glu 20
25 30 Leu Asp Lys Leu Ser Lys Ala Ala Ala
Ser Ser Arg Gln Asn Val Gln 35 40
45 Arg Gly Glu Ile Glu His Lys Ile Ala Leu Thr Leu Val Thr
Ile Leu 50 55 60
Gly Phe Val Thr Arg Phe Trp Gly Ile Ser His Pro Asp Glu Val Val 65
70 75 80 Phe Asp Glu Val His
Phe Gly Lys Phe Ala Ser Tyr Tyr Leu Gln Arg 85
90 95 Thr Tyr Phe Phe Asp Val His Pro Pro Phe
Ala Lys Leu Leu Phe Ala 100 105
110 Phe Val Gly Trp Leu Val Gly Tyr Asp Gly His Phe His Phe Glu
Asn 115 120 125 Ile
Gly Asp Ser Tyr Val Ala Asn Lys Val Pro Tyr Val Ala Phe Arg 130
135 140 Ala Leu Pro Ala Val Leu
Gly Ala Leu Thr Val Ser Val Thr Tyr Leu 145 150
155 160 Ile Met Trp Glu Ser Gly Tyr Ser Leu Pro Ala
Cys Leu Val Ala Thr 165 170
175 Gly Leu Ile Leu Leu Asp Asn Ala His Ile Gly Gln Thr Arg Leu Ile
180 185 190 Leu Leu
Asp Ala Thr Leu Val Leu Ala Met Ala Cys Ser Leu Leu Phe 195
200 205 Tyr Ile Lys Phe Tyr Lys Leu
Arg His Glu Ala Phe Ser Arg Lys Trp 210 215
220 Trp Lys Trp Leu Ile Leu Thr Gly Phe Ala Leu Ser
Cys Asp Ile Ser 225 230 235
240 Thr Lys Tyr Val Gly Leu Phe Ala Phe Val Thr Ile Gly Ser Ala Val
245 250 255 Ile Ile Asp
Leu Trp Asp Leu Leu Asp Ile Lys Arg Arg Asn Gly Ala 260
265 270 Ile Ser Leu Gln Leu Phe Gly Lys
His Phe Ala Ala Arg Ala Ile Gly 275 280
285 Leu Ile Val Leu Pro Phe Leu Phe Tyr Leu Phe Trp Phe
Gln Val His 290 295 300
Phe Ala Val Leu Thr Arg Ser Gly Pro Gly Asp Asp Phe Met Thr Pro 305
310 315 320 Glu Phe Gln Glu
Thr Leu Ser Asp Asn Val Met Leu Ala Asn Ala Val 325
330 335 Asp Ile His Tyr Tyr Asp Tyr Ile Thr
Ile Arg His Lys Glu Thr Lys 340 345
350 Ala Tyr Leu His Ser His Pro Asp Thr Tyr Pro Leu Arg Tyr
Asp Asp 355 360 365
Gly Arg Ile Ser Ser Gln Gly Gln Gln Ile Thr Gly Tyr Pro His Asn 370
375 380 Asp Thr Asn Asn Tyr
Trp Gln Val Leu Pro Ser Asp Asn Val His Asn 385 390
395 400 Thr Glu Arg Ile Val Arg Asn Phe Asp Leu
Val Arg Leu Arg His Ile 405 410
415 Val Thr Asp Lys Ile Leu Leu Ser His Asp Val Ala Ser Pro Tyr
Phe 420 425 430 Pro
Thr Asn Gln Glu Phe Thr Ala Val Thr Ser Glu Glu Ala Phe Gly 435
440 445 Glu Arg Gln Asn Asp Thr
Leu Phe Glu Ile Arg Val Glu Thr Ala Lys 450 455
460 Val Gly Ala Glu Phe Lys Thr Val Ala Ser His
Phe Lys Leu Val His 465 470 475
480 Phe Pro Ser Lys Val Ala Met Trp Thr His Thr Thr Pro Leu Pro Glu
485 490 495 Trp Gly
Tyr Lys Gln Gln Glu Ile Asn Gly Asn Lys Gln Val Thr Val 500
505 510 Ser Ser Asn Met Trp Ile Ala
Glu Asp Ile Pro Ser Leu Pro Gln Asp 515 520
525 Asp Ala Arg Arg Gln Lys Glu Gln Arg Gln Val Lys
Ser Leu Pro Phe 530 535 540
Leu Arg Lys Trp Phe Glu Leu Gln Arg Ser Met Phe Tyr His Asn Asn 545
550 555 560 Lys Leu Thr
Ser Ser His Pro Tyr Ser Ser Gln Pro Tyr His Trp Pro 565
570 575 Phe Leu Leu Arg Gly Val Ser Phe
Trp Thr Gln Asn Asp Thr Arg Gln 580 585
590 Gln Ile Tyr Phe Val Gly Asn Pro Ile Gly Trp Trp Ile
Thr Ser Ser 595 600 605
Leu Leu Ala Val Phe Ala Gly Ile Ile Ala Ala Asp Gln Ile Ser Leu 610
615 620 Arg Arg Asn Ile
Asp Ala Leu Asp His Arg Thr Arg Ser Arg Leu Tyr 625 630
635 640 Asn Ser Thr Gly Phe Phe Trp Leu Ala
Trp Ala Thr His Tyr Phe Pro 645 650
655 Phe Tyr Leu Met Gly Arg Gln Leu Phe Leu His His Tyr Leu
Pro Ala 660 665 670
His Leu Ala Ser Cys Leu Val Thr Gly Ala Leu Val Glu Phe Ile Phe
675 680 685 Asn Ser Asp Ala
Val Glu Glu Glu Ser Ser Lys Ser Gly Asn Arg Ser 690
695 700 Ser Pro Lys Arg His Val Thr Ala
Arg Glu Arg Phe Ala Gly Lys Ser 705 710
715 720 Met Leu Gly Ala Trp Ile Ala Cys Gly Val Ile Leu
Ser Ala Ala Ala 725 730
735 Ala Cys Trp Tyr Phe Phe Leu Pro Leu Thr Tyr Gly Tyr Pro Gly Leu
740 745 750 Ser Val Glu
Glu Val Val Arg Arg Lys Trp Leu Gly Tyr Asp Leu His 755
760 765 Phe Ala Lys 770
10770PRTFusarium oxysporum 10Met Ala Arg Ser Ser Thr Pro Gln Gly Ser Leu
Arg Gln Arg Gly Ala 1 5 10
15 Pro Ser Lys Lys Pro Phe Glu Glu Asp Ser Phe Asp Pro Asn Ile Glu
20 25 30 Leu Asp
Lys Leu Ala Lys Ala Gly Ala Gln Arg Ala Ala Ala Gln Ser 35
40 45 Glu Thr Glu Tyr Lys Ile Gly
Leu Phe Leu Ile Thr Ile Leu Ser Phe 50 55
60 Val Thr Arg Phe Trp Gly Ile Ser His Pro Asn Glu
Val Val Phe Asp 65 70 75
80 Glu Val His Phe Gly Lys Phe Ala Ser Tyr Tyr Leu Glu Arg Thr Tyr
85 90 95 Phe Phe Asp
Val His Pro Pro Phe Gly Lys Leu Leu Phe Ala Phe Val 100
105 110 Gly Trp Leu Val Gly Tyr Asp Gly
Asn Phe His Phe Glu Asn Ile Gly 115 120
125 Asp Ser Tyr Ile Ala Asn Lys Val Pro Tyr Val Ala Tyr
Arg Ala Leu 130 135 140
Pro Ala Thr Leu Gly Ala Leu Thr Val Ser Val Thr Tyr Leu Ile Met 145
150 155 160 Trp Glu Ser Gly
Tyr Ser Leu Pro Ala Cys Ile Leu Ala Ala Gly Leu 165
170 175 Val Leu Leu Asp Asn Ala His Ile Gly
Gln Thr Arg Leu Ile Leu Leu 180 185
190 Asp Ala Thr Leu Val Leu Ala Met Ala Cys Ser Leu Leu Phe
Tyr Ile 195 200 205
Lys Trp Tyr Lys Leu Arg His Glu Pro Phe Ser Arg Lys Trp Trp Lys 210
215 220 Trp Leu Ile Leu Thr
Gly Phe Ala Leu Ser Cys Asp Ile Ser Val Lys 225 230
235 240 Tyr Val Gly Val Phe Ala Phe Val Thr Ile
Gly Ser Ala Val Val Ile 245 250
255 Asp Leu Trp Asp Leu Leu Asn Ile Asn Arg Pro Gly Gly Ala Ile
Ser 260 265 270 Leu
Gln Glu Phe Thr Lys His Phe Ala Ala Arg Ala Phe Gly Leu Ile 275
280 285 Ile Met Pro Phe Leu Phe
Tyr Leu Phe Trp Phe Gln Val His Phe Ala 290 295
300 Val Leu Tyr Arg Ser Gly Pro Gly Asp Asp Phe
Met Thr Pro Glu Phe 305 310 315
320 Gln Glu Thr Leu Ser Asp Asn Val Met Leu Ala Asn Ser Ile Asp Ile
325 330 335 Gln Tyr
Tyr Asp Gln Ile Thr Ile Arg His Lys Glu Thr Lys Thr Tyr 340
345 350 Leu His Ser His Glu Asp Arg
Tyr Pro Leu Arg Tyr Asp Asp Gly Arg 355 360
365 Val Ser Ser Gln Gly Gln Gln Ile Thr Gly Tyr Pro
Tyr Asn Asp Thr 370 375 380
Asn Asn Tyr Trp Glu Ile Leu Pro Ala Asn Asn Asp Lys Gln Ile Gly 385
390 395 400 Arg Ile Val
Lys Asn His Glu Leu Val Arg Leu Arg His Val Gly Thr 405
410 415 Asp Lys Ile Leu Leu Ser His Asp
Val Ala Ser Pro Tyr Tyr Pro Thr 420 425
430 Asn Gln Glu Phe Thr Ala Val Thr Pro Glu Glu Ala Phe
Gly Lys Arg 435 440 445
Glu Lys Asp Thr Leu Phe Glu Val Arg Ile Glu His Gly Lys Lys Asn 450
455 460 Gln Asn Phe Lys
Thr Val Ala Gly His Phe Lys Leu Ile His Asn Pro 465 470
475 480 Ser Lys Val Ala Met Trp Thr His Thr
Lys Pro Leu Pro Glu Trp Gly 485 490
495 Tyr Lys Gln Gln Glu Ile Asn Gly Asn Lys Gln Ile Ala Pro
Ser Ser 500 505 510
Asn Val Trp Ile Ala Glu Asp Ile Pro Ser Leu Pro Ala Asp His Pro
515 520 525 Arg Arg Gln Lys
Pro Glu Arg Lys Val Lys Ser Leu Pro Phe Leu Gln 530
535 540 Lys Trp Phe Glu Leu Gln Arg Ala
Met Phe Tyr His Asn Ser Lys Leu 545 550
555 560 Thr Ser Ser His Pro Tyr Ala Ser His Pro Tyr Gln
Trp Pro Phe Leu 565 570
575 Leu Arg Gly Val Ser Phe Trp Thr Gln Ser Glu Thr Arg Gln Gln Ile
580 585 590 Tyr Phe Leu
Gly Asn Pro Ile Gly Trp Trp Leu Ala Ser Ser Leu Leu 595
600 605 Ala Val Tyr Ala Gly Ile Leu Leu
Ala Asp Gln Val Ser Leu Arg Arg 610 615
620 Gly Val Asp Ala Leu Asp Arg Arg Thr Arg Ser Arg Leu
Tyr Asn Ser 625 630 635
640 Thr Gly Phe Phe Phe Leu Ala Trp Ala Thr His Tyr Phe Pro Phe Phe
645 650 655 Leu Met Gly Arg
Gln Leu Phe Leu His His Tyr Leu Pro Ala His Leu 660
665 670 Ala Ser Cys Leu Val Ala Gly Ala Leu
Leu Glu Phe Ile Phe Asn Ser 675 680
685 Glu Ala Pro Glu Glu Val Thr Ile Lys Asp Lys Lys Gly Pro
Val Ser 690 695 700
Pro Arg His His Val Thr Ala Arg Glu Arg Phe Ala Gly Gln Ser Met 705
710 715 720 Leu Gly Ala Trp Ile
Ala Cys Gly Val Ile Leu Ser Leu Ile Ile Ala 725
730 735 Gly Trp Tyr Phe Phe Leu Pro Leu Thr Tyr
Gly Tyr Pro Gly Leu Ser 740 745
750 Val Asp Ala Ile Leu Arg Arg Lys Trp Leu Gly Tyr Asp Leu His
Phe 755 760 765 Ala
Lys 770 11788PRTGibberella zeae 11Met Ala Arg Ser Ser Ser Pro Ser Gln
Gly Ser Leu Arg Gln Arg Gly 1 5 10
15 Ala Pro Ser Lys Lys Pro Ser Glu Glu Ser Phe Asn Pro Asn
Pro Glu 20 25 30
Leu Asp Lys Leu Ala Lys Ala Gly Ala Gln Arg Ala Ala Ala Gln Ser
35 40 45 Glu Thr Glu His
Lys Ile Gly Leu Ala Val Ile Thr Ile Leu Ser Phe 50
55 60 Val Thr Arg Phe Trp Gly Ile Ser
His Pro Asn Glu Val Val Phe Asp 65 70
75 80 Glu Val His Phe Gly Lys Phe Ala Ser Tyr Tyr Leu
Glu Arg Thr Tyr 85 90
95 Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu Leu Phe Ala Phe Val
100 105 110 Gly Trp Leu
Val Gly Tyr Asp Gly His Phe His Phe Asp Asn Ile Gly 115
120 125 Asp Ser Tyr Ile Ala Asn Lys Ile
Pro Tyr Val Ala Phe Arg Ala Leu 130 135
140 Pro Ala Thr Leu Gly Ala Leu Thr Val Ala Val Thr Tyr
Leu Ile Met 145 150 155
160 Trp Glu Ser Gly Tyr Ser Leu Pro Ala Cys Val Leu Ala Ala Gly Leu
165 170 175 Leu Leu Leu Asp
Asn Ala His Ile Gly Gln Thr Arg Leu Ile Leu Leu 180
185 190 Asp Ala Thr Leu Val Leu Ala Met Ala
Cys Ser Leu Leu Phe Tyr Ile 195 200
205 Lys Trp Tyr Lys Leu Arg His Glu Pro Phe Ser Arg Lys Trp
Trp Lys 210 215 220
Trp Leu Ile Leu Thr Gly Phe Ala Leu Ser Cys Asp Ile Ser Val Lys 225
230 235 240 Tyr Val Gly Val Phe
Ala Phe Val Thr Ile Gly Cys Ala Val Val Ile 245
250 255 Asp Leu Trp Asp Leu Leu Asn Ile Asn Arg
Pro Gly Gly Ala Ile Ser 260 265
270 Met Gln Glu Phe Gly Lys His Phe Ala Ala Arg Ala Phe Gly Leu
Ile 275 280 285 Val
Leu Pro Phe Leu Phe Tyr Leu Phe Trp Phe Gln Val His Phe Ala 290
295 300 Val Leu Tyr Arg Ser Gly
Pro Gly Asp Asp Phe Met Thr Pro Glu Phe 305 310
315 320 Gln Glu Thr Leu Ser Asp Asn Val Met Leu Ala
Asn Ala Ile Asp Ile 325 330
335 Gln Tyr Tyr Asp Ser Ile Thr Ile Arg His Lys Glu Thr Lys Thr Tyr
340 345 350 Leu His
Ser His Glu Asp Arg Tyr Pro Leu Arg Tyr Asp Asp Gly Arg 355
360 365 Val Ser Ser Gln Gly Gln Gln
Ile Thr Gly Tyr Pro Tyr Asn Asp Thr 370 375
380 Asn Asn Tyr Trp Glu Ile Trp Pro Ala Asp Asn Asn
Lys Thr Pro Gly 385 390 395
400 Arg Ile Val Lys Asn His Asp Leu Val Arg Leu Arg His Val Gly Thr
405 410 415 Asp Lys Ile
Leu Leu Ser His Asp Val Ala Ser Pro Tyr Tyr Pro Thr 420
425 430 Asn Gln Glu Phe Thr Ala Val Thr
Pro Glu Glu Ala Leu Gly Lys Arg 435 440
445 Glu Lys Glu Thr Leu Phe Glu Val Arg Leu Glu His Gly
Lys Lys Asn 450 455 460
Gln Asn Phe Lys Ser Val Ala Gly His Phe Lys Leu Ile His Asn Pro 465
470 475 480 Ser Lys Val Ala
Met Trp Thr His Thr Lys Pro Leu Pro Glu Trp Gly 485
490 495 Tyr Lys Gln Gln Glu Ile Asn Gly Asn
Lys Gln Ile Ala Pro Ser Ser 500 505
510 Asn Val Trp Ile Ala Glu Asp Ile Ala Ser Leu Glu Ala Asp
His Pro 515 520 525
Arg Arg Gln Lys Pro Glu Arg Lys Val Lys Ser Leu Pro Phe Leu Gln 530
535 540 Lys Trp Phe Glu Leu
Gln Arg Ala Met Phe Tyr His Asn Ser Lys Leu 545 550
555 560 Thr Ser Ser His Pro Tyr Ala Ser His Pro
Tyr Gln Trp Pro Phe Leu 565 570
575 Leu Arg Gly Val Ser Phe Trp Thr Gln Ser Glu Thr Arg Gln Gln
Ile 580 585 590 Tyr
Phe Leu Gly Asn Pro Val Gly Trp Trp Leu Ala Ser Ser Leu Leu 595
600 605 Ala Val Tyr Ala Gly Ile
Leu Leu Ala Asp Gln Val Ser Leu Arg Arg 610 615
620 Gly Ile Asp Ala Leu Asp Arg Arg Lys Leu Met
Leu Gln Ser Gln Leu 625 630 635
640 Met Asn Pro Thr Leu Thr Asn Ser Lys Gly Thr Arg Ser Arg Leu Tyr
645 650 655 Asn Ser
Thr Gly Phe Phe Phe Leu Ala Trp Ala Thr His Tyr Phe Pro 660
665 670 Phe Phe Leu Met Gly Arg Gln
Leu Phe Leu His His Tyr Leu Pro Ala 675 680
685 His Leu Ala Ser Cys Leu Val Ala Gly Ala Leu Leu
Glu Phe Ile Phe 690 695 700
Asn Ser Glu Pro Ala Glu Glu Ile Thr Ile Lys Asp Lys Lys Gly Pro 705
710 715 720 Val Ser Pro
Arg His His Val Thr Ala Arg Glu Arg Phe Ser Gly Gln 725
730 735 Ser Met Ala Ser Ala Trp Ile Ala
Cys Gly Val Val Leu Ala Leu Val 740 745
750 Val Ala Gly Trp Tyr Phe Phe Leu Pro Leu Thr Tyr Gly
Tyr Pro Gly 755 760 765
Leu Ser Val Glu Ala Ile Leu Arg Arg Lys Trp Leu Gly Tyr Asp Leu 770
775 780 His Phe Ala Lys
785 12775PRTMyceliophthora thermophila 12Met Ala Ser Thr Ser
Thr Pro Gln Gly Thr Leu Arg Gln Arg Asn Val 1 5
10 15 Gly Val Ser Thr Lys Lys Pro Lys Asp Gly
Ala Ser Ser Asp Val Glu 20 25
30 Leu Asp Lys Leu Val Lys Ala Ala Ala Glu Lys Ser Ser Lys Asn
Ser 35 40 45 Glu
Arg Asp Phe Lys Val Val Phe Val Val Met Thr Ala Leu Ala Phe 50
55 60 Leu Thr Arg Phe Trp Gly
Ile Ser His Pro Asn Glu Val Val Phe Asp 65 70
75 80 Glu Val His Phe Gly Lys Phe Ala Ser Tyr Tyr
Leu Glu Arg Thr Tyr 85 90
95 Phe Phe Asp Val His Pro Pro Leu Gly Lys Leu Leu Phe Ala Phe Met
100 105 110 Gly Trp
Leu Val Gly Tyr Asp Gly His Phe His Phe Glu Asn Ile Gly 115
120 125 Asp Ser Tyr Ile Val Asn Lys
Val Pro Tyr Val Ala Phe Arg Ser Leu 130 135
140 Pro Ala Ile Leu Gly Ala Leu Thr Val Ser Val Thr
Tyr Leu Ile Met 145 150 155
160 Trp Glu Ser Gly Tyr Ser Leu Pro Ala Cys Ile Ile Ala Ala Gly Leu
165 170 175 Ile Leu Leu
Asp Asn Ala His Ile Gly Gln Thr Arg Leu Ile Leu Leu 180
185 190 Asp Ala Thr Leu Val Phe Ala Met
Ala Cys Ser Leu Leu Cys Tyr Ile 195 200
205 Lys Phe Tyr Lys Leu Arg His Glu Pro Phe Ser Arg Lys
Trp Trp Lys 210 215 220
Trp Leu Ile Leu Thr Gly Phe Ala Leu Ser Cys Asp Ile Ser Thr Lys 225
230 235 240 Tyr Val Gly Leu
Phe Ala Phe Ile Thr Ile Gly Ser Ala Val Val Ile 245
250 255 Asp Leu Trp Asp Leu Leu Asp Ile Lys
Arg Pro Gly Gly Ala Leu Thr 260 265
270 Leu Ala Glu Phe Gly Lys His Phe Ala Ala Arg Ala Phe Gly
Leu Ile 275 280 285
Ile Met Pro Phe Leu Phe Tyr Leu Phe Trp Phe Gln Val His Phe Ser 290
295 300 Ile Leu Thr Arg Ser
Gly Pro Gly Asp Asp Phe Met Thr Pro Glu Phe 305 310
315 320 Gln Glu Thr Leu Ser Asp Asn Ile Met Leu
Ala Asn Ala Val Thr Ile 325 330
335 Asp Tyr Tyr Asp Thr Ile Leu Ile Lys His Lys Glu Thr Lys Val
Tyr 340 345 350 Leu
His Ser His Pro Asp Arg Tyr Pro Leu Arg Tyr Asp Asp Gly Arg 355
360 365 Val Ser Ser Gln Gly Gln
Gln Val Thr Gly Tyr Pro Phe Asn Asp Thr 370 375
380 Asn Asn Tyr Trp Gln Ile Leu Pro Gly Gly Ala
Asp Asp Gln Lys Leu 385 390 395
400 Gly Arg His Val Arg Asn His Asp Leu Val Arg Leu Arg His Leu Gly
405 410 415 Thr Asp
Thr Ile Leu Leu Ser His Asp Val Ala Ser Pro Tyr Tyr Pro 420
425 430 Thr Asn Gln Glu Phe Thr Thr
Val Ser Ile Ala Asp Ala Tyr Gly Glu 435 440
445 Arg Ala Ala Asp Thr Leu Phe Glu Ile Arg Ile Glu
His Gly Lys Asp 450 455 460
Gly Gln Glu Phe Lys Ser Val Ser Ser His Phe Lys Leu Ile His Asn 465
470 475 480 Pro Ser Lys
Val Ala Met Trp Thr His Pro Lys Pro Leu Pro Asp Trp 485
490 495 Gly Tyr Lys Gln Gln Glu Ile Asn
Gly Asn Lys Gln Ile Ala Pro Ser 500 505
510 Ser Asn Val Trp Leu Val Glu Asp Ile Val Ser Leu Pro
Pro Asp His 515 520 525
Lys Arg Arg Glu Lys Pro Glu Arg Lys Val Lys Thr Leu Pro Phe Leu 530
535 540 Arg Lys Trp Phe
Glu Leu Gln Arg Ser Met Phe Trp His Asn Asn Gln 545 550
555 560 Leu Thr Ala Ser His Pro Tyr Ala Ser
Leu Pro Tyr Gln Trp Pro Phe 565 570
575 Leu Leu Arg Gly Val Ser Phe Trp Thr Gln Asn Glu Thr Arg
Gln Gln 580 585 590
Ile Tyr Phe Leu Gly Asn Pro Val Gly Trp Trp Ile Ala Ser Ser Val
595 600 605 Leu Ala Ile Tyr
Ala Gly Ile Val Leu Ala Asp Gln Phe Ser Leu Arg 610
615 620 Arg Gly Ile Asp Ala Leu Asp His
Arg Ser Arg Ser Arg Leu Tyr Asn 625 630
635 640 Ser Thr Gly Phe Phe Phe Leu Ala Trp Ala Thr His
Tyr Phe Pro Phe 645 650
655 Tyr Val Met Gly Arg Gln Leu Phe Leu His His Tyr Leu Pro Ala His
660 665 670 Leu Ala Ser
Ala Leu Val Thr Gly Ala Ile Val Glu Phe Ile Phe Ala 675
680 685 Gln Asp Ser Leu Glu His Glu Val
Ala Tyr Gln Ala Ala Lys Ala Gly 690 695
700 Lys Lys Thr Gly Val Gln Lys Arg His Leu Ser Ala Arg
Glu Arg Phe 705 710 715
720 Ala Gly Gln Ser Met Val Ala Ser Trp Ile Ala Thr Val Val Ile Leu
725 730 735 Ile Ala Val Ala
Ala Ser Trp Tyr Phe Phe Leu Pro Leu Thr Tyr Gly 740
745 750 Tyr Pro Gly Leu Ser Val Asp Gln Val
Leu Arg Arg Lys Trp Leu Gly 755 760
765 Tyr Asp Leu His Phe Ala Lys 770 775
13774PRTNeurospora crassa 13Met Ala Ser Thr Thr Ala Thr Pro Glu Ala Thr
Leu Arg Gln Arg Asn 1 5 10
15 Val Pro Ala Ser Ser Lys Lys Ala Lys Asn Gly Val Ser Ser Asp Val
20 25 30 Glu Thr
Asp Lys Val Pro Asp Ala Val Ala Pro Ala Lys Ser Gly Ser 35
40 45 Glu Leu Glu Tyr Lys Leu Ala
Leu Ile Leu Ile Thr Gly Leu Ala Phe 50 55
60 Leu Thr Arg Phe Trp Gly Ile Ser His Pro Asp Glu
Val Val Phe Asp 65 70 75
80 Glu Val His Phe Gly Lys Phe Ala Ser Tyr Tyr Leu Glu Arg Thr Tyr
85 90 95 Phe Phe Asp
Val His Pro Pro Phe Gly Lys Leu Leu Phe Ala Phe Met 100
105 110 Gly Trp Leu Val Gly Tyr Asp Gly
His Phe His Phe Glu Asn Ile Gly 115 120
125 Asp Ser Tyr Ile Arg Asn Lys Val Pro Tyr Val Ala Phe
Arg Ser Leu 130 135 140
Pro Ala Ile Leu Gly Ala Leu Thr Val Ser Val Val Tyr Met Ile Met 145
150 155 160 Trp Glu Ser Gly
Tyr Ser Leu Pro Ala Cys Leu Ile Ala Ala Gly Leu 165
170 175 Val Leu Leu Asp Asn Ala His Ile Gly
Gln Thr Arg Leu Ile Leu Leu 180 185
190 Asp Ala Thr Leu Val Phe Ala Met Ala Cys Ser Leu Leu Cys
Tyr Ile 195 200 205
Lys Phe His Lys Leu Arg His Glu Pro Phe Ser Arg Lys Trp Trp Lys 210
215 220 Trp Leu Ile Leu Thr
Gly Phe Ala Leu Ser Cys Asp Ile Ser Thr Lys 225 230
235 240 Tyr Val Gly Leu Phe Ala Phe Ile Thr Ile
Gly Ser Ala Val Cys Ile 245 250
255 Asp Leu Trp Asp Leu Leu Asp Ile Lys Arg Pro Gly Gly Ala Leu
Thr 260 265 270 Leu
Pro Gln Phe Gly Lys His Phe Ala Ala Arg Ala Phe Gly Leu Ile 275
280 285 Ile Met Pro Phe Ile Phe
Tyr Leu Phe Trp Phe Gln Val His Phe Ser 290 295
300 Ile Leu Thr Arg Ser Gly Pro Gly Asp Asp Phe
Met Thr Pro Glu Phe 305 310 315
320 Gln Glu Thr Leu Ser Asp Asn Ile Met Leu Ala Asn Ala Val Thr Ile
325 330 335 Asp Tyr
Tyr Asp Thr Ile Ser Ile Arg His Lys Glu Thr Lys Ala Tyr 340
345 350 Leu His Ser His Pro Asp Lys
Tyr Pro Leu Arg Tyr Asp Asp Gly Arg 355 360
365 Val Ser Ser Gln Gly Gln Gln Val Thr Gly Tyr Pro
Phe Asn Asp Thr 370 375 380
Asn Asn Tyr Trp Gln Ile Leu Pro Pro Gly Pro Asp Asp Gln Lys Leu 385
390 395 400 Gly His Pro
Ile Lys Asn His Asp Leu Val Arg Leu Arg His Ile Val 405
410 415 Thr Asp Thr Ile Leu Leu Ser His
Asp Val Ala Ser Pro Tyr Tyr Pro 420 425
430 Thr Asn Gln Glu Phe Thr Thr Val Ser Ile Gly Asp Ala
Tyr Gly Asp 435 440 445
Arg Ala Ala Asp Thr Leu Phe Glu Ile Arg Ile Glu His Gly Lys Ala 450
455 460 Asn Gln Glu Phe
Lys Ser Ile Ser Ser His Phe Lys Leu Ile His Asn 465 470
475 480 Pro Ser Lys Val Ala Met Trp Thr His
Ser Lys Pro Leu Pro Glu Trp 485 490
495 Gly His Lys Gln Gln Glu Ile Asn Gly Asn Lys Gln Leu Ala
Gln Ser 500 505 510
Ser Asn Val Trp Leu Val Glu Asp Ile Val Ser Leu Pro Ala Asp His
515 520 525 Ala Arg Arg Glu
Lys Pro Glu Lys Lys Val Lys Thr Leu Pro Phe Leu 530
535 540 Arg Lys Trp Phe Glu Leu Gln Arg
Ser Met Phe Trp His Asn Asn Gln 545 550
555 560 Leu Thr Ser Ser His Pro Tyr Ala Ser Leu Pro Tyr
Gln Trp Pro Phe 565 570
575 Leu Leu Arg Gly Val Ser Phe Trp Thr Gln Asn Asp Thr Arg Gln Gln
580 585 590 Ile Tyr Phe
Leu Gly Asn Pro Ile Gly Trp Trp Leu Ala Ser Ser Val 595
600 605 Leu Ala Ile Tyr Ala Gly Ile Ile
Leu Ala Asp Gln Phe Ser Leu Arg 610 615
620 Arg Gly Leu Asp Ala Met Asp Arg Arg Thr Arg Ser Arg
Leu Tyr Asn 625 630 635
640 Ser Thr Gly Phe Phe Phe Leu Ala Trp Ala Thr His Tyr Phe Pro Phe
645 650 655 Phe Val Met Gly
Arg Gln Leu Phe Leu His His Tyr Leu Pro Ala His 660
665 670 Leu Ala Ser Ala Leu Val Thr Gly Ser
Val Val Glu Phe Leu Phe Ser 675 680
685 Thr Asp Ser Ala Glu Pro Glu Tyr Gln Pro Ser Lys Ser Gly
Lys Lys 690 695 700
Val Ala Pro Thr Thr Lys Arg Arg Leu Ser Ala Arg Glu Arg Leu Ala 705
710 715 720 Gly Gln Ser Met Ala
Gly Ala Trp Ile Ala Thr Ala Val Ile Met Val 725
730 735 Leu Val Ala Phe Gly Trp Tyr Phe Phe Leu
Pro Leu Thr Tyr Gly Tyr 740 745
750 Pro Gly Leu Thr Ala Pro Glu Val Asn Arg Arg Lys Trp Leu Gly
Tyr 755 760 765 Asp
Leu His Phe Ala Lys 770 14776PRTPenicillium
chrysogenum 14Met Ser Ser Pro Ser Pro Ser Leu Arg Lys Arg Gly Gly Lys Lys
Asp 1 5 10 15 Val
Tyr Thr Ala Leu Pro Ser Asp Asp Thr Ser Thr Pro Val Ser Val
20 25 30 Pro Val Lys Gln Lys
Ser Glu Trp Asp Tyr Trp Leu Ala Ile Val Ile 35
40 45 Leu Thr Leu Leu Ala Phe Ala Thr Arg
Phe Tyr Arg Leu Asp Tyr Pro 50 55
60 Asn Glu Val Val Phe Asp Glu Val His Phe Gly Lys Phe
Ala Ser Tyr 65 70 75
80 Tyr Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys
85 90 95 Leu Leu Phe Ala
Leu Met Gly Trp Leu Val Gly Phe Asp Gly Ser Phe 100
105 110 Leu Phe Glu Asn Ile Gly Asp Ser Tyr
Ile Glu Asn Asn Val Pro Tyr 115 120
125 Leu Ser Leu Arg Ala Met Pro Ala Thr Leu Gly Ala Leu Thr
Ile Pro 130 135 140
Val Val Phe Leu Ile Met Trp Glu Ser Gly Tyr Ser Leu Pro Ala Cys 145
150 155 160 Val Leu Ser Ala Gly
Leu Met Val Phe Asp Asn Ala His Val Gly Glu 165
170 175 Asp Arg Leu Ile Leu Leu Asp Ala Thr Leu
Val Leu Ser Met Ala Leu 180 185
190 Ser Ile Leu Cys Tyr Val Arg Phe Tyr Lys Leu Arg His Gln Pro
Phe 195 200 205 Gly
Arg Lys Trp Trp Lys Trp Leu Leu Leu Thr Gly Phe Cys Met Ser 210
215 220 Cys Val Ile Ser Thr Lys
Tyr Val Gly Phe Phe Thr Phe Val Thr Ile 225 230
235 240 Gly Ala Ala Val Leu Ile Asp Leu Trp Asn Leu
Leu Asp Ile Asn Arg 245 250
255 Glu Gln Gly Ala Leu Ser Met Ile Ser Trp Gly Lys His Phe Ile Ala
260 265 270 Arg Ala
Val Gly Leu Val Ile Ile Pro Phe Met Phe Tyr Leu Phe Trp 275
280 285 Phe Gln Val His Phe Ala Ile
Leu Asn Arg Ser Gly Pro Gly Asp Asp 290 295
300 Phe Met Thr Pro Glu Phe Gln Glu Thr Leu Ser Asp
Asn Gln Met Thr 305 310 315
320 Ala Gln Ser Val Gly Ile Gln Tyr Phe Asp Thr Ile Thr Met Arg His
325 330 335 Lys Asp Thr
Lys Val Phe Leu His Ser His Trp Asp Lys Tyr Pro Leu 340
345 350 Arg Tyr Asp Asp Gly Arg Ile Ser
Ser Gln Gly Gln Gln Val Thr Gly 355 360
365 Tyr Pro His Asn Asp Thr Asn Asn Gln Trp Gln Ile Leu
Pro Ala Glu 370 375 380
Pro Leu Ala Asp Ser Ser Glu Pro Lys Ser Val Arg Asn Gly Asp Ile 385
390 395 400 Ile Gln Leu Arg
His Ile Gly Thr Glu Ser Tyr Leu Leu Thr His Asp 405
410 415 Val Ala Ser Pro Phe Phe Pro Thr Asn
Gln Glu Phe Thr Thr Val Ser 420 425
430 Gln Glu Leu Ala Asp Gly Glu Arg His Asn Asp Thr Leu Phe
Glu Leu 435 440 445
Lys Ile Glu Ser Gly Lys Thr Ala Gln Glu Phe Arg Thr Leu Ala Ser 450
455 460 Leu Phe Lys Leu Val
His Val Pro Thr Arg Val Ala Leu Trp Thr His 465 470
475 480 Thr Thr Pro Leu Pro Glu Trp Gly Tyr Lys
Gln Ala Glu Ile Asn Gly 485 490
495 Asn Lys Asn Ile Leu Gln Ser Ser Asn Met Trp Tyr Val Glu Asn
Ile 500 505 510 Glu
Asn Leu Ala Glu Asp Ser Pro Arg Leu Val Lys Glu Glu Arg Lys 515
520 525 Val Lys Thr Leu Pro Phe
Leu Arg Lys Tyr Phe Glu Leu Gln Gly Ala 530 535
540 Met Phe His His Asn Asn Ala Leu Thr Ser Ser
His Pro Tyr Ala Thr 545 550 555
560 Glu Pro Phe Gln Trp Pro Phe Leu Leu Arg Gly Val Ser Phe Trp Thr
565 570 575 Lys Asn
Asp Thr Arg Glu Gln Ile Tyr Phe Leu Gly Asn Pro Ile Gly 580
585 590 Trp Trp Ile Ala Ser Ser Ile
Leu Ala Val Phe Ala Gly Val Val Gly 595 600
605 Ala Asp Gln Leu Ser Leu Arg Arg Gly Val Asp Ala
Leu Glu Glu Ile 610 615 620
Trp Gly Pro Gly Thr Arg Ser Arg Leu Tyr Asn Ser Thr Gly Phe Leu 625
630 635 640 Phe Leu Cys
Trp Ala Ala His Tyr Phe Pro Phe Trp Leu Met Gly Arg 645
650 655 Gln Arg Phe Leu His His Tyr Leu
Pro Ser His Leu Ala Ser Thr Met 660 665
670 Val Cys Gly Ala Leu Ile Glu Phe Ile Phe Asn Leu Gln
Pro Leu Asp 675 680 685
Pro Arg Thr Ala Leu Pro Pro Val Asp Asp Pro Ser Gly Lys Ser Lys 690
695 700 Ala Arg Ser Leu
Ser Ser Leu Arg Arg Phe Ile Thr Ala Lys Glu Arg 705 710
715 720 Met Gly Cys Arg Ser Leu Ile Ala Gly
Trp Ile Ala Thr Leu Ile Ile 725 730
735 Leu Ala Ala Thr Ile Trp Gly Phe Ile Phe Tyr Ala Pro Leu
Thr Tyr 740 745 750
Gly Thr Pro Gly Leu Asp Val Ala Gly Val Asn Ala Arg Lys Trp Leu
755 760 765 Asn Tyr Asp Leu
His Phe Ala Lys 770 775 1519DNATrichoderma reesei
15gcacactttc aagattggc
191619DNATrichoderma reesei 16gtacggtgtt gccaagaag
1917448DNATrichoderma reesei 17gttgagtaca
tcgagcgcga cagcattgtg cacaccatgc ttcccctcga gtccaaggac 60agcatcatcg
ttgaggactc gtgcaacggc gagacggaga agcaggctcc ctggggtctt 120gcccgtatct
ctcaccgaga gacgctcaac tttggctcct tcaacaagta cctctacacc 180gctgatggtg
gtgagggtgt tgatgcctat gtcattgaca ccggcaccaa catcgagcac 240gtcgactttg
agggtcgtgc caagtggggc aagaccatcc ctgccggcga tgaggacgag 300gacggcaacg
gccacggcac tcactgctct ggtaccgttg ctggtaagaa gtacggtgtt 360gccaagaagg
cccacgtcta cgccgtcaag gtgctccgat ccaacggatc cggcaccatg 420tctgacgtcg
tcaagggcgt cgagtacg
44818399PRTTrichoderma reesei 18Met Gln Pro Ser Phe Gly Ser Phe Leu Val
Thr Val Leu Ser Ala Ser 1 5 10
15 Met Ala Ala Gly Ser Val Ile Pro Ser Thr Asn Ala Asn Pro Gly
Ser 20 25 30 Phe
Glu Ile Lys Arg Ser Ala Asn Lys Ala Phe Thr Gly Arg Asn Gly 35
40 45 Pro Leu Ala Leu Ala Arg
Thr Tyr Ala Lys Tyr Gly Val Glu Val Pro 50 55
60 Lys Thr Leu Val Asp Ala Ile Gln Leu Val Lys
Ser Ile Gln Leu Ala 65 70 75
80 Lys Arg Asp Ser Ala Thr Val Thr Ala Thr Pro Asp His Asp Asp Ile
85 90 95 Glu Tyr
Leu Val Pro Val Lys Ile Gly Thr Pro Pro Gln Thr Leu Asn 100
105 110 Leu Asp Phe Asp Thr Gly Ser
Ser Asp Leu Trp Val Phe Ser Ser Asp 115 120
125 Val Asp Pro Thr Ser Ser Gln Gly His Asp Ile Tyr
Thr Pro Ser Lys 130 135 140
Ser Thr Ser Ser Lys Lys Leu Glu Gly Ala Ser Trp Asn Ile Thr Tyr 145
150 155 160 Gly Asp Arg
Ser Ser Ser Ser Gly Asp Val Tyr His Asp Ile Val Ser 165
170 175 Val Gly Asn Leu Thr Val Lys Ser
Gln Ala Val Glu Ser Ala Arg Asn 180 185
190 Val Ser Ala Gln Phe Thr Gln Gly Asn Asn Asp Gly Leu
Val Gly Leu 195 200 205
Ala Phe Ser Ser Ile Asn Thr Val Lys Pro Thr Pro Gln Lys Thr Trp 210
215 220 Tyr Asp Asn Ile
Val Gly Ser Leu Asp Ser Pro Val Phe Val Ala Asp 225 230
235 240 Leu Arg His Asp Thr Pro Gly Ser Tyr
His Phe Gly Ser Ile Pro Ser 245 250
255 Glu Ala Ser Lys Ala Phe Tyr Ala Pro Ile Asp Asn Ser Lys
Gly Phe 260 265 270
Trp Gln Phe Ser Thr Ser Ser Asn Ile Ser Gly Gln Phe Asn Ala Val
275 280 285 Ala Asp Thr Gly
Thr Thr Leu Leu Leu Ala Ser Asp Asp Leu Val Lys 290
295 300 Ala Tyr Tyr Ala Lys Val Gln Gly
Ala Arg Val Asn Val Phe Leu Gly 305 310
315 320 Gly Tyr Val Phe Asn Cys Thr Thr Gln Leu Pro Asp
Phe Thr Phe Thr 325 330
335 Val Gly Glu Gly Asn Ile Thr Val Pro Gly Thr Leu Ile Asn Tyr Ser
340 345 350 Glu Ala Gly
Asn Gly Gln Cys Phe Gly Gly Ile Gln Pro Ser Gly Gly 355
360 365 Leu Pro Phe Ala Ile Phe Gly Asp
Ile Ala Leu Lys Ala Ala Tyr Val 370 375
380 Ile Phe Asp Ser Gly Asn Lys Gln Val Gly Trp Ala Gln
Lys Lys 385 390 395
19452PRTTrichoderma reesei 19Met Glu Ala Ile Leu Gln Ala Gln Ala Lys Phe
Arg Leu Asp Arg Gly 1 5 10
15 Leu Gln Lys Ile Thr Ala Val Arg Asn Lys Asn Tyr Lys Arg His Gly
20 25 30 Pro Lys
Ser Tyr Val Tyr Leu Leu Asn Arg Phe Gly Phe Glu Pro Thr 35
40 45 Lys Pro Gly Pro Tyr Phe Gln
Gln His Arg Ile His Gln Arg Gly Leu 50 55
60 Ala His Pro Asp Phe Lys Ala Ala Val Gly Gly Arg
Val Thr Arg Gln 65 70 75
80 Lys Val Leu Ala Lys Lys Val Lys Glu Asp Gly Thr Val Asp Ala Gly
85 90 95 Gly Ser Lys
Thr Gly Glu Val Asp Ala Glu Asp Gln Gln Asn Asp Ser 100
105 110 Glu Tyr Leu Cys Glu Val Thr Ile
Gly Thr Pro Gly Gln Lys Leu Met 115 120
125 Leu Asp Phe Asp Thr Gly Ser Ser Asp Leu Trp Val Phe
Ser Thr Glu 130 135 140
Leu Ser Lys His Leu Gln Glu Asn His Ala Ile Phe Asp Pro Lys Lys 145
150 155 160 Ser Ser Thr Phe
Lys Pro Leu Lys Asp Gln Thr Trp Gln Ile Ser Tyr 165
170 175 Gly Asp Gly Ser Ser Ala Ser Gly Thr
Cys Gly Ser Asp Thr Val Thr 180 185
190 Leu Gly Gly Leu Ser Ile Lys Asn Gln Thr Ile Glu Leu Ala
Ser Lys 195 200 205
Leu Ala Pro Gln Phe Ala Gln Gly Thr Gly Asp Gly Leu Leu Gly Leu 210
215 220 Ala Trp Pro Gln Ile
Asn Thr Val Gln Thr Asp Gly Arg Pro Thr Pro 225 230
235 240 Ala Asn Thr Pro Val Ala Asn Met Ile Gln
Gln Asp Asp Ile Pro Ser 245 250
255 Asp Ala Gln Leu Phe Thr Ala Ala Phe Tyr Ser Glu Arg Asp Glu
Asn 260 265 270 Ala
Glu Ser Phe Tyr Thr Phe Gly Tyr Ile Asp Gln Asp Leu Val Ser 275
280 285 Ala Ser Gly Gln Glu Ile
Ala Trp Thr Asp Val Asp Asn Ser Gln Gly 290 295
300 Phe Trp Met Phe Pro Ser Thr Lys Thr Thr Ile
Asn Gly Lys Asp Ile 305 310 315
320 Ser Gln Glu Gly Asn Thr Ala Ile Ala Asp Thr Gly Thr Thr Leu Ala
325 330 335 Leu Val
Ser Asp Glu Val Cys Glu Ala Leu Tyr Lys Ala Ile Pro Gly 340
345 350 Ala Lys Tyr Asp Asp Asn Gln
Gln Gly Tyr Val Phe Pro Ile Asn Thr 355 360
365 Asp Ala Ser Ser Leu Pro Glu Leu Lys Val Ser Val
Gly Asn Thr Gln 370 375 380
Phe Val Ile Gln Pro Glu Asp Leu Ala Phe Ala Pro Ala Asp Asp Ser 385
390 395 400 Asn Trp Tyr
Gly Gly Val Gln Ser Arg Gly Ser Asn Pro Phe Asp Ile 405
410 415 Leu Gly Asp Val Phe Leu Lys Ser
Val Tyr Ala Ile Phe Asp Gln Gly 420 425
430 Asn Gln Arg Phe Gly Ala Val Pro Lys Ile Gln Ala Lys
Gln Asn Leu 435 440 445
Gln Pro Pro Gln 450 20395PRTTrichoderma reesei 20Met Lys
Ser Ala Leu Leu Ala Ala Ala Ala Leu Val Gly Ser Ala Gln 1 5
10 15 Ala Gly Ile His Lys Met Lys
Leu Gln Lys Val Ser Leu Glu Gln Gln 20 25
30 Leu Glu Gly Ser Ser Ile Glu Ala His Val Gln Gln
Leu Gly Gln Lys 35 40 45
Tyr Met Gly Val Arg Pro Thr Ser Arg Ala Glu Val Met Phe Asn Asp
50 55 60 Lys Pro Pro
Lys Val Gln Gly Gly His Pro Val Pro Val Thr Asn Phe 65
70 75 80 Met Asn Ala Gln Tyr Phe Ser
Glu Ile Thr Ile Gly Thr Pro Pro Gln 85
90 95 Ser Phe Lys Val Val Leu Asp Thr Gly Ser Ser
Asn Leu Trp Val Pro 100 105
110 Ser Gln Ser Cys Asn Ser Ile Ala Cys Phe Leu His Ser Thr Tyr
Asp 115 120 125 Ser
Ser Ser Ser Ser Thr Tyr Lys Pro Asn Gly Ser Asp Phe Glu Ile 130
135 140 His Tyr Gly Ser Gly Ser
Leu Thr Gly Phe Ile Ser Asn Asp Val Val 145 150
155 160 Thr Ile Gly Asp Leu Lys Ile Lys Gly Gln Asp
Phe Ala Glu Ala Thr 165 170
175 Ser Glu Pro Gly Leu Ala Phe Ala Phe Gly Arg Phe Asp Gly Ile Leu
180 185 190 Gly Leu
Gly Tyr Asp Thr Ile Ser Val Asn Gly Ile Val Pro Pro Phe 195
200 205 Tyr Gln Met Val Asn Gln Lys
Leu Ile Asp Glu Pro Val Phe Ala Phe 210 215
220 Tyr Leu Gly Ser Ser Asp Glu Gly Ser Glu Ala Val
Phe Gly Gly Val 225 230 235
240 Asp Asp Ala His Tyr Glu Gly Lys Ile Glu Tyr Ile Pro Leu Arg Arg
245 250 255 Lys Ala Tyr
Trp Glu Val Asp Leu Asp Ser Ile Ala Phe Gly Asp Glu 260
265 270 Val Ala Glu Leu Glu Asn Thr Gly
Ala Ile Leu Asp Thr Gly Thr Ser 275 280
285 Leu Asn Val Leu Pro Ser Gly Leu Ala Glu Leu Leu Asn
Ala Glu Ile 290 295 300
Gly Ala Lys Lys Gly Phe Gly Gly Gln Tyr Thr Val Asp Cys Ser Lys 305
310 315 320 Arg Asp Ser Leu
Pro Asp Ile Thr Phe Ser Leu Ala Gly Ser Lys Tyr 325
330 335 Ser Leu Pro Ala Ser Asp Tyr Ile Ile
Glu Met Ser Gly Asn Cys Ile 340 345
350 Ser Ser Phe Gln Gly Met Asp Phe Pro Glu Pro Val Gly Pro
Leu Val 355 360 365
Ile Leu Gly Asp Ala Phe Leu Arg Arg Tyr Tyr Ser Val Tyr Asp Leu 370
375 380 Gly Arg Asp Ala Val
Gly Leu Ala Lys Ala Lys 385 390 395
21426PRTTrichoderma reesei 21Met Lys Phe His Ala Ala Ala Leu Thr Leu Ala
Cys Leu Ala Ser Ser 1 5 10
15 Ala Ser Ala Gly Val Ala Gln Pro Arg Ala Asp Glu Val Glu Ser Ala
20 25 30 Glu Gln
Gly Lys Thr Phe Ser Leu Glu Gln Ile Pro Asn Glu Arg Tyr 35
40 45 Lys Gly Asn Ile Pro Ala Ala
Tyr Ile Ser Ala Leu Ala Lys Tyr Ser 50 55
60 Pro Thr Ile Pro Asp Lys Ile Lys His Ala Ile
Glu Ile Asn Pro Asp 65 70 75
80 Leu His Arg Lys Phe Ser Lys Leu Ile Asn Ala Gly Asn Met Thr Gly
85 90 95 Thr Ala
Val Ala Ser Pro Pro Pro Gly Ala Asp Ala Glu Tyr Val Leu 100
105 110 Pro Val Lys Ile Gly Thr Pro
Pro Gln Thr Leu Pro Leu Asn Leu Asp 115 120
125 Thr Gly Ser Ser Asp Leu Trp Val Ile Ser Thr Asp
Thr Tyr Pro Pro 130 135 140
Gln Val Gln Gly Gln Thr Arg Tyr Asn Val Ser Ala Ser Thr Thr Ala 145
150 155 160 Gln Arg Leu
Ile Gly Glu Ser Trp Val Ile Arg Tyr Gly Asp Gly Ser 165
170 175 Ser Ala Asn Gly Ile Val Tyr Lys
Asp Arg Val Gln Ile Gly Asn Thr 180 185
190 Phe Phe Asn Gln Gln Ala Val Glu Ser Ala Val Asn Ile
Ser Asn Glu 195 200 205
Ile Ser Asp Asp Ser Phe Ser Ser Gly Leu Leu Gly Ala Ala Ser Ser 210
215 220 Ala Ala Asn Thr
Val Arg Pro Asp Arg Gln Thr Thr Tyr Leu Glu Asn 225 230
235 240 Ile Lys Ser Gln Leu Ala Arg Pro Val
Phe Thr Ala Asn Leu Lys Lys 245 250
255 Gly Lys Pro Gly Asn Tyr Asn Phe Gly Tyr Ile Asn Gly Ser
Glu Tyr 260 265 270
Ile Gly Pro Ile Gln Tyr Ala Ala Ile Asn Pro Ser Ser Pro Leu Trp
275 280 285 Glu Val Ser Val
Ser Gly Tyr Arg Val Gly Ser Asn Asp Thr Lys Tyr 290
295 300 Val Pro Arg Val Trp Asn Ala Ile
Ala Asp Thr Gly Thr Thr Leu Leu 305 310
315 320 Leu Val Pro Asn Asp Ile Val Ser Ala Tyr Tyr Ala
Gln Val Lys Gly 325 330
335 Ser Thr Phe Ser Asn Asp Val Gly Met Met Leu Val Pro Cys Ala Ala
340 345 350 Thr Leu Pro
Asp Phe Ala Phe Gly Leu Gly Asn Tyr Arg Gly Val Ile 355
360 365 Pro Gly Ser Tyr Ile Asn Tyr Gly
Arg Met Asn Lys Thr Tyr Cys Tyr 370 375
380 Gly Gly Ile Gln Ser Ser Glu Asp Ala Pro Phe Ala Val
Leu Gly Asp 385 390 395
400 Ile Ala Leu Lys Ala Gln Phe Val Val Phe Asp Met Gly Asn Lys Val
405 410 415 Val Gly Phe Ala
Asn Lys Asn Thr Asn Val 420 425
22407PRTTrichoderma reesei 22Met Gln Thr Phe Gly Ala Phe Leu Val Ser Phe
Leu Ala Ala Ser Gly 1 5 10
15 Leu Ala Ala Ala Leu Pro Thr Glu Gly Gln Lys Thr Ala Ser Val Glu
20 25 30 Val Gln
Tyr Asn Lys Asn Tyr Val Pro His Gly Pro Thr Ala Leu Phe 35
40 45 Lys Ala Lys Arg Lys Tyr Gly
Ala Pro Ile Ser Asp Asn Leu Lys Ser 50 55
60 Leu Val Ala Ala Arg Gln Ala Lys Gln Ala Leu Ala
Lys Arg Gln Thr 65 70 75
80 Gly Ser Ala Pro Asn His Pro Ser Asp Ser Ala Asp Ser Glu Tyr Ile
85 90 95 Thr Ser Val
Ser Ile Gly Thr Pro Ala Gln Val Leu Pro Leu Asp Phe 100
105 110 Asp Thr Gly Ser Ser Asp Leu Trp
Val Phe Ser Ser Glu Thr Pro Lys 115 120
125 Ser Ser Ala Thr Gly His Ala Ile Tyr Thr Pro Ser Lys
Ser Ser Thr 130 135 140
Ser Lys Lys Val Ser Gly Ala Ser Trp Ser Ile Ser Tyr Gly Asp Gly 145
150 155 160 Ser Ser Ser Ser
Gly Asp Val Tyr Thr Asp Lys Val Thr Ile Gly Gly 165
170 175 Phe Ser Val Asn Thr Gln Gly Val Glu
Ser Ala Thr Arg Val Ser Thr 180 185
190 Glu Phe Val Gln Asp Thr Val Ile Ser Gly Leu Val Gly Leu
Ala Phe 195 200 205
Asp Ser Gly Asn Gln Val Arg Pro His Pro Gln Lys Thr Trp Phe Ser 210
215 220 Asn Ala Ala Ser Ser
Leu Ala Glu Pro Leu Phe Thr Ala Asp Leu Arg 225 230
235 240 His Gly Gln Asn Gly Ser Tyr Asn Phe Gly
Tyr Ile Asp Thr Ser Val 245 250
255 Ala Lys Gly Pro Val Ala Tyr Thr Pro Val Asp Asn Ser Gln Gly
Phe 260 265 270 Trp
Glu Phe Thr Ala Ser Gly Tyr Ser Val Gly Gly Gly Lys Leu Asn 275
280 285 Arg Asn Ser Ile Asp Gly
Ile Ala Asp Thr Gly Thr Thr Leu Leu Leu 290 295
300 Leu Asp Asp Asn Val Val Asp Ala Tyr Tyr Ala
Asn Val Gln Ser Ala 305 310 315
320 Gln Tyr Asp Asn Gln Gln Glu Gly Val Val Phe Asp Cys Asp Glu Asp
325 330 335 Leu Pro
Ser Phe Ser Phe Gly Val Gly Ser Ser Thr Ile Thr Ile Pro 340
345 350 Gly Asp Leu Leu Asn Leu Thr
Pro Leu Glu Glu Gly Ser Ser Thr Cys 355 360
365 Phe Gly Gly Leu Gln Ser Ser Ser Gly Ile Gly Ile
Asn Ile Phe Gly 370 375 380
Asp Val Ala Leu Lys Ala Ala Leu Val Val Phe Asp Leu Gly Asn Glu 385
390 395 400 Arg Leu Gly
Trp Ala Gln Lys 405 23446PRTTrichoderma reesei
23Met Thr Leu Pro Val Pro Leu Arg Glu His Asp Leu Pro Phe Leu Lys 1
5 10 15 Glu Lys Arg Lys
Leu Pro Ala Asp Asp Ile Pro Ser Gly Thr Tyr Thr 20
25 30 Leu Pro Ile Ile His Ala Arg Arg Pro
Lys Leu Ala Ser Arg Ala Ile 35 40
45 Glu Val Gln Val Glu Asn Arg Ser Asp Val Ser Tyr Tyr Ala
Gln Leu 50 55 60
Asn Ile Gly Thr Pro Pro Gln Thr Val Tyr Ala Gln Ile Asp Thr Gly 65
70 75 80 Ser Phe Glu Leu Trp
Val Asn Pro Asn Cys Ser Asn Val Gln Ser Ala 85
90 95 Asp Gln Arg Phe Cys Arg Ala Ile Gly Phe
Tyr Asp Pro Ser Ser Ser 100 105
110 Ser Thr Ala Asp Val Thr Ser Gln Ser Ala Arg Leu Arg Tyr Gly
Ile 115 120 125 Gly
Ser Ala Asp Val Thr Tyr Val His Asp Thr Ile Ser Leu Pro Gly 130
135 140 Ser Gly Ser Gly Ser Lys
Ala Met Lys Ala Val Gln Phe Gly Val Ala 145 150
155 160 Asp Thr Ser Val Asp Glu Phe Ser Gly Ile Leu
Gly Leu Gly Ala Gly 165 170
175 Asn Gly Ile Asn Thr Glu Tyr Pro Asn Phe Val Asp Glu Leu Ala Ala
180 185 190 Gln Gly
Val Thr Ala Thr Lys Ala Phe Ser Leu Ala Leu Gly Ser Lys 195
200 205 Ala Glu Glu Glu Gly Val Ile
Ile Phe Gly Gly Val Asp Thr Ala Lys 210 215
220 Phe His Gly Glu Leu Ala His Leu Pro Ile Val Pro
Ala Asp Asp Ser 225 230 235
240 Pro Asp Gly Val Ala Arg Tyr Trp Val Lys Met Lys Ser Ile Ser Leu
245 250 255 Thr Pro Pro
Pro Pro Ser Ser Ser Gly Ser Thr Asp Asp Asn Asn Asn 260
265 270 Lys Pro Val Ala Phe Pro Gln Thr
Ser Met Thr Val Phe Leu Asp Ser 275 280
285 Gly Ser Thr Leu Thr Leu Leu Pro Pro Ala Leu Val Arg
Gln Ile Ala 290 295 300
Ser Ala Leu Gly Ser Thr Gln Thr Asp Glu Ser Gly Phe Phe Val Val 305
310 315 320 Asp Cys Ala Leu
Ala Ser Gln Asp Gly Thr Ile Asp Phe Glu Phe Asp 325
330 335 Gly Val Thr Ile Arg Val Pro Tyr Ala
Glu Met Ile Arg Gln Val Ser 340 345
350 Thr Leu Pro Pro His Cys Tyr Leu Gly Met Met Gly Ser Thr
Gln Phe 355 360 365
Ala Leu Leu Gly Asp Thr Phe Leu Arg Ser Ala Tyr Ala Val Phe Asp 370
375 380 Leu Thr Ser Asn Val
Val His Leu Ala Pro Tyr Ala Asn Cys Gly Thr 385 390
395 400 Asn Val Lys Ser Ile Thr Ser Thr Ser Ser
Leu Ser Asn Leu Val Gly 405 410
415 Thr Cys Asn Asp Pro Ser Lys Pro Ser Ser Ser Pro Ser Pro Ser
Gln 420 425 430 Thr
Pro Ser Ala Ser Pro Ser Ser Thr Ala Thr Gln Lys Ala 435
440 445 24259PRTTrichoderma reesei 24Met Ala
Pro Ala Ser Gln Val Val Ser Ala Leu Met Leu Pro Ala Leu 1 5
10 15 Ala Leu Gly Ala Ala Ile Gln
Pro Arg Gly Ala Asp Ile Val Gly Gly 20 25
30 Thr Ala Ala Ser Leu Gly Glu Phe Pro Tyr Ile Val
Ser Leu Gln Asn 35 40 45
Pro Asn Gln Gly Gly His Phe Cys Gly Gly Val Leu Val Asn Ala Asn
50 55 60 Thr Val Val
Thr Ala Ala His Cys Ser Val Val Tyr Pro Ala Ser Gln 65
70 75 80 Ile Arg Val Arg Ala Gly Thr
Leu Thr Trp Asn Ser Gly Gly Thr Leu 85
90 95 Val Gly Val Ser Gln Ile Ile Val Asn Pro Ser
Tyr Asn Asp Arg Thr 100 105
110 Thr Asp Phe Asp Val Ala Val Trp His Leu Ser Ser Pro Ile Arg
Glu 115 120 125 Ser
Ser Thr Ile Gly Tyr Ala Thr Leu Pro Ala Gln Gly Ser Asp Pro 130
135 140 Val Ala Gly Ser Thr Val
Thr Thr Ala Gly Trp Gly Thr Thr Ser Glu 145 150
155 160 Asn Ser Asn Ser Ile Pro Ser Arg Leu Asn Lys
Val Ser Val Pro Val 165 170
175 Val Ala Arg Ser Thr Cys Gln Ala Asp Tyr Arg Ser Gln Gly Leu Ser
180 185 190 Val Thr
Asn Asn Met Phe Cys Ala Gly Leu Thr Gln Gly Gly Lys Asp 195
200 205 Ser Cys Ser Gly Asp Ser Gly
Gly Pro Ile Val Asp Ala Asn Gly Val 210 215
220 Leu Gln Gly Val Val Ser Trp Gly Ile Gly Cys Ala
Glu Ala Gly Phe 225 230 235
240 Pro Gly Val Tyr Thr Arg Ile Gly Asn Phe Val Asn Tyr Ile Asn Gln
245 250 255 Asn Leu Ala
25882PRTTrichoderma reesei 25Met Val Arg Ser Ala Leu Phe Val Ser Leu Leu
Ala Thr Phe Ser Gly 1 5 10
15 Val Ile Ala Arg Val Ser Gly His Gly Ser Lys Ile Val Pro Gly Ala
20 25 30 Tyr Ile
Phe Glu Phe Glu Asp Ser Gln Asp Thr Ala Asp Phe Tyr Lys 35
40 45 Lys Leu Asn Gly Glu Gly Ser
Thr Arg Leu Lys Phe Asp Tyr Lys Leu 50 55
60 Phe Lys Gly Val Ser Val Gln Leu Lys Asp Leu Asp
Asn His Glu Ala 65 70 75
80 Lys Ala Gln Gln Met Ala Gln Leu Pro Ala Val Lys Asn Val Trp Pro
85 90 95 Val Thr Leu
Ile Asp Ala Pro Asn Pro Lys Val Glu Trp Val Ala Gly 100
105 110 Ser Thr Ala Pro Thr Leu Glu Ser
Arg Ala Ile Lys Lys Pro Pro Ile 115 120
125 Pro Asn Asp Ser Ser Asp Phe Pro Thr His Gln Met Thr
Gln Ile Asp 130 135 140
Lys Leu Arg Ala Lys Gly Tyr Thr Gly Lys Gly Val Arg Val Ala Val 145
150 155 160 Ile Asp Thr Gly
Ile Asp Tyr Thr His Pro Ala Leu Gly Gly Cys Phe 165
170 175 Gly Arg Gly Cys Leu Val Ser Phe Gly
Thr Asp Leu Val Gly Asp Asp 180 185
190 Tyr Thr Gly Phe Asn Thr Pro Val Pro Asp Asp Asp Pro Val
Asp Cys 195 200 205
Ala Gly His Gly Ser His Val Ala Gly Ile Ile Ala Ala Gln Glu Asn 210
215 220 Pro Tyr Gly Phe Thr
Gly Gly Ala Pro Asp Val Thr Leu Gly Ala Tyr 225 230
235 240 Arg Val Phe Gly Cys Asp Gly Gln Ala Gly
Asn Asp Val Leu Ile Ser 245 250
255 Ala Tyr Asn Gln Ala Phe Glu Asp Gly Ala Gln Ile Ile Thr Ala
Ser 260 265 270 Ile
Gly Gly Pro Ser Gly Trp Ala Glu Glu Pro Trp Ala Val Ala Val 275
280 285 Thr Arg Ile Val Glu Ala
Gly Val Pro Cys Thr Val Ser Ala Gly Asn 290 295
300 Glu Gly Asp Ser Gly Leu Phe Phe Ala Ser Thr
Ala Ala Asn Gly Lys 305 310 315
320 Lys Val Ile Ala Val Ala Ser Val Asp Asn Glu Asn Ile Pro Ser Val
325 330 335 Leu Ser
Val Ala Ser Tyr Lys Ile Asp Ser Gly Ala Ala Gln Asp Phe 340
345 350 Gly Tyr Val Ser Ser Ser Lys
Ala Trp Asp Gly Val Ser Lys Pro Leu 355 360
365 Tyr Ala Val Ser Phe Asp Thr Thr Ile Pro Asp Asp
Gly Cys Ser Pro 370 375 380
Leu Pro Asp Ser Thr Pro Asp Leu Ser Asp Tyr Ile Val Leu Val Arg 385
390 395 400 Arg Gly Thr
Cys Thr Phe Val Gln Lys Ala Gln Asn Val Ala Ala Lys 405
410 415 Gly Ala Lys Tyr Leu Leu Tyr Tyr
Asn Asn Ile Pro Gly Ala Leu Ala 420 425
430 Val Asp Val Ser Ala Val Pro Glu Ile Glu Ala Val Gly
Met Val Asp 435 440 445
Asp Lys Thr Gly Ala Thr Trp Ile Ala Ala Leu Lys Asp Gly Lys Thr 450
455 460 Val Thr Leu Thr
Leu Thr Asp Pro Ile Glu Ser Glu Lys Gln Ile Gln 465 470
475 480 Phe Ser Asp Asn Pro Thr Thr Gly Gly
Ala Leu Ser Gly Tyr Thr Thr 485 490
495 Trp Gly Pro Thr Trp Glu Leu Asp Val Lys Pro Gln Ile Ser
Ser Pro 500 505 510
Gly Gly Asn Ile Leu Ser Thr Tyr Pro Val Ala Leu Gly Gly Tyr Ala
515 520 525 Thr Leu Ser Gly
Thr Ser Met Ala Cys Pro Leu Thr Ala Ala Ala Val 530
535 540 Ala Leu Ile Gly Gln Ala Arg Gly
Thr Phe Asp Pro Ala Leu Ile Asp 545 550
555 560 Asn Leu Leu Ala Thr Thr Ala Asn Pro Gln Leu Phe
Asn Asp Gly Glu 565 570
575 Lys Phe Tyr Asp Phe Leu Ala Pro Val Pro Gln Gln Gly Gly Gly Leu
580 585 590 Ile Gln Ala
Tyr Asp Ala Ala Phe Ala Thr Thr Leu Leu Ser Pro Ser 595
600 605 Ser Leu Ser Phe Asn Asp Thr Asp
His Phe Ile Lys Lys Lys Gln Ile 610 615
620 Thr Leu Lys Asn Thr Ser Lys Gln Arg Val Thr Tyr Lys
Leu Asn His 625 630 635
640 Val Pro Thr Asn Thr Phe Tyr Thr Leu Ala Pro Gly Asn Gly Tyr Pro
645 650 655 Ala Pro Phe Pro
Asn Asp Ala Val Ala Ala His Ala Asn Leu Lys Phe 660
665 670 Asn Leu Gln Gln Val Thr Leu Pro Ala
Gly Arg Ser Ile Thr Val Asp 675 680
685 Val Phe Pro Thr Pro Pro Arg Asp Val Asp Ala Lys Arg Leu
Ala Leu 690 695 700
Trp Ser Gly Tyr Ile Thr Val Asn Gly Thr Asp Gly Thr Ser Leu Ser 705
710 715 720 Val Pro Tyr Gln Gly
Leu Thr Gly Ser Leu His Lys Gln Lys Val Leu 725
730 735 Tyr Pro Glu Asp Ser Trp Ile Ala Asp Ser
Thr Asp Glu Ser Leu Ala 740 745
750 Pro Val Glu Asn Gly Thr Val Phe Thr Ile Pro Ala Pro Gly Asn
Ala 755 760 765 Gly
Pro Asp Asp Lys Leu Pro Ser Leu Val Val Ser Pro Ala Leu Gly 770
775 780 Ser Arg Tyr Val Arg Val
Asp Leu Val Leu Leu Ser Ala Pro Pro His 785 790
795 800 Gly Thr Lys Leu Lys Thr Val Lys Phe Leu Asp
Thr Thr Ser Ile Gly 805 810
815 Gln Pro Ala Gly Ser Pro Leu Leu Trp Ile Ser Arg Gly Ala Asn Pro
820 825 830 Ile Ala
Trp Thr Gly Glu Leu Ser Asp Asn Lys Phe Ala Pro Pro Gly 835
840 845 Thr Tyr Lys Ala Val Phe His
Ala Leu Arg Ile Phe Gly Asn Glu Lys 850 855
860 Lys Lys Glu Asp Trp Asp Val Ser Glu Ser Pro Ala
Phe Thr Ile Lys 865 870 875
880 Tyr Ala 26541PRTTrichoderma reesei 26Met Arg Ser Val Val Ala Leu
Ser Met Ala Ala Val Ala Gln Ala Ser 1 5
10 15 Thr Phe Gln Ile Gly Thr Ile His Glu Lys Ser
Ala Pro Val Leu Ser 20 25
30 Asn Val Glu Ala Asn Ala Ile Pro Asp Ala Tyr Ile Ile Lys Phe
Lys 35 40 45 Asp
His Val Gly Glu Asp Asp Ala Ser Lys His His Asp Trp Ile Gln 50
55 60 Ser Ile His Thr Asn Val
Glu Gln Glu Arg Leu Glu Leu Arg Lys Arg 65 70
75 80 Ser Asn Val Phe Gly Ala Asp Asp Val Phe Asp
Gly Leu Lys His Thr 85 90
95 Phe Lys Ile Gly Asp Gly Phe Lys Gly Tyr Ala Gly His Phe His Glu
100 105 110 Ser Val
Ile Glu Gln Val Arg Asn His Pro Asp Val Glu Tyr Ile Glu 115
120 125 Arg Asp Ser Ile Val His Thr
Met Leu Pro Leu Glu Ser Lys Asp Ser 130 135
140 Ile Ile Val Glu Asp Ser Cys Asn Gly Glu Thr Glu
Lys Gln Ala Pro 145 150 155
160 Trp Gly Leu Ala Arg Ile Ser His Arg Glu Thr Leu Asn Phe Gly Ser
165 170 175 Phe Asn Lys
Tyr Leu Tyr Thr Ala Asp Gly Gly Glu Gly Val Asp Ala 180
185 190 Tyr Val Ile Asp Thr Gly Thr Asn
Ile Glu His Val Asp Phe Glu Gly 195 200
205 Arg Ala Lys Trp Gly Lys Thr Ile Pro Ala Gly Asp Glu
Asp Glu Asp 210 215 220
Gly Asn Gly His Gly Thr His Cys Ser Gly Thr Val Ala Gly Lys Lys 225
230 235 240 Tyr Gly Val Ala
Lys Lys Ala His Val Tyr Ala Val Lys Val Leu Arg 245
250 255 Ser Asn Gly Ser Gly Thr Met Ser Asp
Val Val Lys Gly Val Glu Tyr 260 265
270 Ala Ala Leu Ser His Ile Glu Gln Val Lys Lys Ala Lys Lys
Gly Lys 275 280 285
Arg Lys Gly Phe Lys Gly Ser Val Ala Asn Met Ser Leu Gly Gly Gly 290
295 300 Lys Thr Gln Ala Leu
Asp Ala Ala Val Asn Ala Ala Val Arg Ala Gly 305 310
315 320 Val His Phe Ala Val Ala Ala Gly Asn Asp
Asn Ala Asp Ala Cys Asn 325 330
335 Tyr Ser Pro Ala Ala Ala Thr Glu Pro Leu Thr Val Gly Ala Ser
Ala 340 345 350 Leu
Asp Asp Ser Arg Ala Tyr Phe Ser Asn Tyr Gly Lys Cys Thr Asp 355
360 365 Ile Phe Ala Pro Gly Leu
Ser Ile Gln Ser Thr Trp Ile Gly Ser Lys 370 375
380 Tyr Ala Val Asn Thr Ile Ser Gly Thr Ser Met
Ala Ser Pro His Ile 385 390 395
400 Cys Gly Leu Leu Ala Tyr Tyr Leu Ser Leu Gln Pro Ala Gly Asp Ser
405 410 415 Glu Phe
Ala Val Ala Pro Ile Thr Pro Lys Lys Leu Lys Glu Ser Val 420
425 430 Ile Ser Val Ala Thr Lys Asn
Ala Leu Ser Asp Leu Pro Asp Ser Asp 435 440
445 Thr Pro Asn Leu Leu Ala Trp Asn Gly Gly Gly Cys
Ser Asn Phe Ser 450 455 460
Gln Ile Val Glu Ala Gly Ser Tyr Thr Val Lys Pro Lys Gln Asn Lys 465
470 475 480 Gln Ala Lys
Leu Pro Ser Thr Ile Glu Glu Leu Glu Glu Ala Ile Glu 485
490 495 Gly Asp Phe Glu Val Val Ser Gly
Glu Ile Val Lys Gly Ala Lys Ser 500 505
510 Phe Gly Ser Lys Ala Glu Lys Phe Ala Lys Lys Ile His
Asp Leu Val 515 520 525
Glu Glu Glu Ile Glu Glu Phe Ile Ser Glu Leu Ser Glu 530
535 540 27391PRTTrichoderma reesei 27Met Arg Leu
Ser Val Leu Leu Ser Val Leu Pro Leu Val Leu Ala Ala 1 5
10 15 Pro Ala Ile Glu Lys Arg Ala Glu
Pro Ala Pro Leu Leu Val Pro Thr 20 25
30 Thr Lys His Gly Leu Val Ala Asp Lys Tyr Ile Val Lys
Phe Lys Asp 35 40 45
Gly Ser Ser Leu Gln Ala Val Asp Glu Ala Ile Ser Gly Leu Val Ser 50
55 60 Asn Ala Asp His
Val Tyr Gln His Val Phe Arg Gly Phe Ala Ala Thr 65 70
75 80 Leu Asp Lys Glu Thr Leu Glu Ala Leu
Arg Asn His Pro Glu Val Asp 85 90
95 Tyr Ile Glu Gln Asp Ala Val Val Lys Ile Asn Ala Tyr Val
Ser Gln 100 105 110
Thr Gly Ala Pro Trp Gly Leu Gly Arg Ile Ser His Lys Ala Arg Gly
115 120 125 Ser Thr Thr Tyr
Val Tyr Asp Asp Ser Ala Gly Ala Gly Thr Cys Ser 130
135 140 Tyr Val Ile Asp Thr Gly Val Asp
Ala Thr His Pro Asp Phe Glu Gly 145 150
155 160 Arg Ala Thr Leu Leu Arg Ser Phe Val Ser Gly Gln
Asn Thr Asp Gly 165 170
175 Asn Gly His Gly Thr His Val Ser Gly Thr Ile Gly Ser Arg Thr Tyr
180 185 190 Gly Val Ala
Lys Lys Thr Gln Ile Tyr Gly Val Lys Val Leu Asp Asn 195
200 205 Ser Gly Ser Gly Ser Phe Ser Thr
Val Ile Ala Gly Met Asp Tyr Val 210 215
220 Ala Ser Asp Ser Gln Thr Arg Asn Cys Pro Asn Gly Ser
Val Ala Asn 225 230 235
240 Met Ser Leu Gly Gly Gly Tyr Thr Ala Ser Val Asn Gln Ala Ala Ala
245 250 255 Arg Leu Ile Gln
Ala Gly Val Phe Leu Ala Val Ala Ala Gly Asn Asp 260
265 270 Gly Val Asp Ala Arg Asn Thr Ser Pro
Ala Ser Glu Pro Thr Val Cys 275 280
285 Thr Val Gly Ala Ser Thr Ser Ser Asp Ala Arg Ala Ser Phe
Ser Asn 290 295 300
Tyr Gly Ser Val Val Asp Ile Phe Ala Pro Gly Gln Asp Ile Leu Ser 305
310 315 320 Thr Trp Pro Asn Arg
Gln Thr Asn Thr Ile Ser Gly Thr Ser Met Ala 325
330 335 Thr Pro His Ile Val Gly Leu Gly Ala Tyr
Leu Ala Gly Leu Glu Gly 340 345
350 Phe Ser Asp Pro Gln Ala Leu Cys Ala Arg Ile Gln Ser Leu Ala
Asn 355 360 365 Arg
Asn Leu Leu Ser Gly Ile Pro Ser Gly Thr Ile Asn Ala Ile Ala 370
375 380 Phe Asn Gly Asn Pro Ser
Gly 385 390 28387PRTTricochoderma reesei 28Met Gly
Leu Val Thr Asn Pro Phe Ala Lys Asn Ile Ile Pro Asn Arg 1 5
10 15 Tyr Ile Val Val Tyr Asn Asn
Ser Phe Gly Glu Glu Ala Ile Ser Ala 20 25
30 Lys Gln Ala Gln Phe Ala Ala Lys Ile Ala Lys Arg
Asn Leu Gly Lys 35 40 45
Arg Gly Leu Phe Gly Asn Glu Leu Ser Thr Ala Ile His Ser Phe Ser
50 55 60 Met His Thr
Trp Arg Ala Met Ala Leu Asp Ala Asp Asp Ile Met Ile 65
70 75 80 Lys Asp Ile Phe Asp Ala Glu
Glu Val Ala Tyr Ile Glu Ala Asp Thr 85
90 95 Lys Val Gln His Ala Ala Leu Val Ala Gln Thr
Asn Ala Ala Pro Gly 100 105
110 Leu Ile Arg Leu Ser Asn Lys Ala Val Gly Gly Gln Asn Tyr Ile
Phe 115 120 125 Asp
Asn Ser Ala Gly Ser Asn Ile Thr Ala Tyr Val Val Asp Thr Gly 130
135 140 Ile Arg Ile Thr His Ser
Glu Phe Glu Gly Arg Ala Thr Phe Gly Ala 145 150
155 160 Asn Phe Val Asn Asp Asp Thr Asp Glu Asn Gly
His Gly Ser His Val 165 170
175 Ala Gly Thr Ile Gly Gly Ala Thr Phe Gly Val Ala Lys Asn Val Glu
180 185 190 Leu Val
Ala Val Lys Val Leu Asp Ala Asp Gly Ser Gly Ser Asn Ser 195
200 205 Gly Val Leu Asn Gly Met Gln
Phe Val Val Asn Asp Val Gln Ala Lys 210 215
220 Lys Arg Ser Gly Lys Ala Val Met Asn Met Ser Leu
Gly Gly Ser Phe 225 230 235
240 Ser Thr Ala Val Asn Asn Ala Ile Thr Ala Leu Thr Asn Ala Gly Ile
245 250 255 Val Pro Val
Val Ala Ala Gly Asn Glu Asn Gln Asp Thr Ala Asn Thr 260
265 270 Ser Pro Gly Ser Ala Pro Gln Ala
Ile Thr Val Gly Ala Ile Asp Ala 275 280
285 Thr Thr Asp Ile Arg Ala Gly Phe Ser Asn Phe Gly Thr
Gly Val Asp 290 295 300
Ile Tyr Ala Pro Gly Val Asp Val Leu Ser Val Gly Ile Lys Ser Asp 305
310 315 320 Ile Asp Thr Ala
Val Leu Ser Gly Thr Ser Met Ala Ser Pro His Val 325
330 335 Ala Gly Leu Ala Ala Tyr Leu Met Ala
Leu Glu Gly Val Ser Asn Val 340 345
350 Asp Asp Val Ser Asn Leu Ile Lys Asn Leu Ala Ala Lys Thr
Gly Ala 355 360 365
Ala Val Lys Gln Asn Ile Ala Gly Thr Thr Ser Leu Ile Ala Asn Asn 370
375 380 Gly Asn Phe 385
29409PRTTrichoderma reesei 29Met Ala Ser Leu Arg Arg Leu Ala Leu Tyr
Leu Gly Ala Leu Leu Pro 1 5 10
15 Ala Val Leu Ala Ala Pro Ala Val Asn Tyr Lys Leu Pro Glu Ala
Val 20 25 30 Pro
Asn Lys Phe Ile Val Thr Leu Lys Asp Gly Ala Ser Val Asp Thr 35
40 45 Asp Ser His Leu Thr Trp
Val Lys Asp Leu His Arg Arg Ser Leu Gly 50 55
60 Lys Arg Ser Thr Ala Gly Val Glu Lys Thr Tyr
Asn Ile Asp Ser Trp 65 70 75
80 Asn Ala Tyr Ala Gly Glu Phe Asp Glu Glu Thr Val Lys Gln Ile Lys
85 90 95 Ala Asn
Pro Asp Val Ala Ser Val Glu Pro Asp Tyr Ile Met Trp Leu 100
105 110 Ser Asp Ile Val Glu Asp Lys
Arg Ala Leu Thr Thr Gln Thr Gly Ala 115 120
125 Pro Trp Gly Leu Gly Thr Val Ser His Arg Thr Pro
Gly Ser Thr Ser 130 135 140
Tyr Ile Tyr Asp Thr Ser Ala Gly Ser Gly Thr Phe Ala Tyr Val Val 145
150 155 160 Asp Ser Gly
Ile Asn Ile Ala His Gln Gln Phe Gly Gly Arg Ala Ser 165
170 175 Leu Gly Tyr Asn Ala Ala Gly Gly
Asp His Val Asp Thr Leu Gly His 180 185
190 Gly Thr His Val Ser Gly Thr Ile Gly Gly Ser Thr Tyr
Gly Val Ala 195 200 205
Lys Gln Ala Ser Leu Ile Ser Val Lys Val Phe Gln Gly Asn Ser Ala 210
215 220 Ser Thr Ser Val
Ile Leu Asp Gly Tyr Asn Trp Ala Val Asn Asp Ile 225 230
235 240 Val Ser Arg Asn Arg Ala Ser Lys Ser
Ala Ile Asn Met Ser Leu Gly 245 250
255 Gly Pro Ala Ser Ser Thr Trp Ala Thr Ala Ile Asn Ala Ala
Phe Asn 260 265 270
Lys Gly Val Leu Thr Ile Val Ala Ala Gly Asn Gly Asp Ala Leu Gly
275 280 285 Asn Pro Gln Pro
Val Ser Ser Thr Ser Pro Ala Asn Val Pro Asn Ala 290
295 300 Ile Thr Val Ala Ala Leu Asp Ile
Asn Trp Arg Thr Ala Ser Phe Thr 305 310
315 320 Asn Tyr Gly Ala Gly Val Asp Val Phe Ala Pro Gly
Val Asn Ile Leu 325 330
335 Ser Ser Trp Ile Gly Ser Asn Thr Ala Thr Asn Thr Ile Ser Gly Thr
340 345 350 Ser Met Ala
Thr Pro His Val Val Gly Leu Ala Leu Tyr Leu Gln Ala 355
360 365 Leu Glu Gly Leu Ser Thr Pro Thr
Ala Val Thr Asn Arg Ile Lys Ala 370 375
380 Leu Ala Thr Thr Gly Arg Val Thr Gly Ser Leu Asn Gly
Ser Pro Asn 385 390 395
400 Thr Leu Ile Phe Asn Gly Asn Ser Ala 405
30555PRTTrichoderma reesei 30Met Arg Ala Cys Leu Leu Phe Leu Gly Ile
Thr Ala Leu Ala Thr Ala 1 5 10
15 Ile Pro Ala Leu Lys Pro Pro His Gly Ser Pro Asp Arg Ala His
Thr 20 25 30 Thr
Gln Leu Ala Lys Val Ser Ile Ala Leu Gln Pro Glu Cys Arg Glu 35
40 45 Leu Leu Glu Gln Ala Leu
His His Leu Ser Asp Pro Ser Ser Pro Arg 50 55
60 Tyr Gly Arg Tyr Leu Gly Arg Glu Glu Ala Lys
Ala Leu Leu Arg Pro 65 70 75
80 Arg Arg Glu Ala Thr Ala Ala Val Lys Arg Trp Leu Ala Arg Ala Gly
85 90 95 Val Pro
Ala His Asp Val Leu Thr Asp Gly Gln Phe Ile His Val Arg 100
105 110 Thr Leu Ala Glu Lys Ala Gln
Ala Leu Leu Gly Phe Glu Tyr Asn Ser 115 120
125 Thr Leu Gly Ser Gln Thr Ile Ala Ile Ser Thr Leu
Pro Gly Lys Ile 130 135 140
Arg Lys His Val Met Thr Val Gln Tyr Val Pro Leu Trp Thr Glu Ala 145
150 155 160 Asp Trp Glu
Glu Cys Lys Thr Ile Ile Thr Pro Ser Cys Leu Lys Arg 165
170 175 Leu Tyr His Val Asp Ser Tyr Arg
Ala Lys Tyr Glu Ser Ser Ser Leu 180 185
190 Phe Gly Ile Val Gly Phe Ser Gly Gln Ala Ala Gln His
Asp Glu Leu 195 200 205
Asp Lys Phe Leu His Asp Phe Ala Pro Tyr Ser Thr Asn Ala Asn Phe 210
215 220 Ser Ile Glu Ser
Val Asn Gly Gly Gln Ser Pro Gln Gly Met Asn Glu 225 230
235 240 Pro Ala Ser Glu Ala Asn Gly Asp Val
Gln Tyr Ala Val Ala Met Gly 245 250
255 Tyr His Val Pro Val Arg Tyr Tyr Ala Val Gly Gly Glu Asn
His Asp 260 265 270
Ile Ile Pro Asp Leu Asp Leu Val Asp Thr Thr Glu Glu Tyr Leu Glu
275 280 285 Pro Phe Leu Glu
Phe Ala Ser His Leu Leu Asp Leu Asp Asp Asp Glu 290
295 300 Leu Pro Arg Val Val Ser Ile Ser
Tyr Gly Ala Asn Glu Gln Leu Phe 305 310
315 320 Pro Arg Ser Tyr Ala His Gln Val Cys Asp Met Phe
Gly Gln Leu Gly 325 330
335 Ala Arg Gly Val Ser Ile Val Val Ala Ala Gly Asp Leu Gly Pro Gly
340 345 350 Val Ser Cys
Gln Ser Asn Asp Gly Ser Ala Arg Pro Lys Phe Ile Pro 355
360 365 Ser Phe Pro Ala Thr Cys Pro Tyr
Val Thr Ser Val Gly Ser Thr Arg 370 375
380 Gly Ile Met Pro Glu Val Ala Ala Ser Phe Ser Ser Gly
Gly Phe Ser 385 390 395
400 Asp Tyr Phe Ala Arg Pro Ala Trp Gln Asp Arg Ala Val Gly Ala Tyr
405 410 415 Leu Gly Ala His
Gly Glu Glu Trp Glu Gly Phe Tyr Asn Pro Ala Gly 420
425 430 Arg Gly Phe Pro Asp Val Ala Ala Gln
Gly Val Asn Phe Arg Phe Arg 435 440
445 Ala His Gly Asn Glu Ser Leu Ser Ser Gly Thr Ser Leu Ser
Ser Pro 450 455 460
Val Phe Ala Ala Leu Ile Ala Leu Leu Asn Asp His Arg Ser Lys Ser 465
470 475 480 Gly Met Pro Pro Met
Gly Phe Leu Asn Pro Trp Ile Tyr Thr Val Gly 485
490 495 Ser His Ala Phe Thr Asp Ile Ile Glu Ala
Arg Ser Glu Gly Cys Pro 500 505
510 Gly Gln Ser Val Glu Tyr Leu Ala Ser Pro Tyr Ile Pro Asn Ala
Gly 515 520 525 Trp
Ser Ala Val Pro Gly Trp Asp Pro Val Thr Gly Trp Gly Thr Pro 530
535 540 Leu Phe Asp Arg Met Leu
Asn Leu Ser Leu Val 545 550 555
31388PRTTrichoderma reesei 31Met Ala Trp Leu Lys Lys Leu Ala Leu Val Leu
Leu Ala Ile Val Pro 1 5 10
15 Tyr Ala Thr Ala Ser Pro Ala Leu Ser Pro Arg Ser Arg Glu Ile Leu
20 25 30 Ser Leu
Glu Asp Leu Glu Ser Glu Asp Lys Tyr Val Ile Gly Leu Lys 35
40 45 Gln Gly Leu Ser Pro Thr Asp
Leu Lys Lys His Leu Leu Arg Val Ser 50 55
60 Ala Val Gln Tyr Arg Asn Lys Asn Ser Thr Phe Glu
Gly Gly Thr Gly 65 70 75
80 Val Lys Arg Thr Tyr Ala Ile Gly Asp Tyr Arg Ala Tyr Thr Ala Val
85 90 95 Leu Asp Arg
Asp Thr Val Arg Glu Ile Trp Asn Asp Thr Leu Glu Lys 100
105 110 Pro Pro Trp Gly Leu Ala Thr Leu
Ser Asn Lys Lys Pro His Gly Phe 115 120
125 Leu Tyr Arg Tyr Asp Lys Ser Ala Gly Glu Gly Thr Phe
Ala Tyr Val 130 135 140
Leu Asp Thr Gly Ile Asn Ser Lys His Val Asp Phe Glu Gly Arg Ala 145
150 155 160 Tyr Met Gly Phe
Ser Pro Pro Lys Thr Glu Pro Thr Asp Ile Asn Gly 165
170 175 His Gly Thr His Val Ala Gly Ile Ile
Gly Gly Lys Thr Phe Gly Val 180 185
190 Ala Lys Lys Thr Gln Leu Ile Gly Val Lys Val Phe Leu Asp
Asp Glu 195 200 205
Ala Thr Thr Ser Thr Leu Met Glu Gly Leu Glu Trp Ala Val Asn Asp 210
215 220 Ile Thr Thr Lys Gly
Arg Gln Gly Arg Ser Val Ile Asn Met Ser Leu 225 230
235 240 Gly Gly Pro Tyr Ser Gln Ala Leu Asn Asp
Ala Ile Asp His Ile Ala 245 250
255 Asp Met Gly Ile Leu Pro Val Ala Ala Ala Gly Asn Lys Gly Ile
Pro 260 265 270 Ala
Thr Phe Ile Ser Pro Ala Ser Ala Asp Lys Ala Met Thr Val Gly 275
280 285 Ala Ile Asn Ser Asp Trp
Gln Glu Thr Asn Phe Ser Asn Phe Gly Pro 290 295
300 Gln Val Asn Ile Leu Ala Pro Gly Glu Asp Val
Leu Ser Ala Tyr Val 305 310 315
320 Ser Thr Asn Thr Ala Thr Arg Val Leu Ser Gly Thr Ser Met Ala Ala
325 330 335 Pro His
Val Ala Gly Leu Ala Leu Tyr Leu Met Ala Leu Glu Glu Phe 340
345 350 Asp Ser Thr Gln Lys Leu Thr
Asp Arg Ile Leu Gln Leu Gly Met Lys 355 360
365 Asn Lys Val Val Asn Leu Met Thr Asp Ser Pro Asn
Leu Ile Ile His 370 375 380
Asn Asn Val Lys 385 32256PRTTrichoderma reesei 32Met
Phe Ile Ala Gly Val Ala Leu Ser Ala Leu Leu Cys Ala Asp Thr 1
5 10 15 Val Leu Ala Gly Val Ala
Gln Asp Arg Gly Leu Ala Ala Arg Leu Ala 20
25 30 Arg Arg Ala Gly Arg Arg Ser Ala Pro Phe
Arg Asn Asp Thr Ser His 35 40
45 Ala Thr Val Gln Ser Asn Trp Gly Gly Ala Ile Leu Glu Gly
Ser Gly 50 55 60
Phe Thr Ala Ala Ser Ala Thr Val Asn Val Pro Arg Gly Gly Gly Gly 65
70 75 80 Ser Asn Ala Ala Gly
Ser Ala Trp Val Gly Ile Asp Gly Ala Ser Cys 85
90 95 Gln Thr Ala Ile Leu Gln Thr Gly Phe Asp
Trp Tyr Gly Asp Gly Thr 100 105
110 Tyr Asp Ala Trp Tyr Glu Trp Tyr Pro Glu Phe Ala Ala Asp Phe
Ser 115 120 125 Gly
Ile Asp Ile Arg Gln Gly Asp Gln Ile Ala Met Ser Val Val Ala 130
135 140 Thr Ser Leu Thr Gly Gly
Ser Ala Thr Leu Glu Asn Leu Ser Thr Gly 145 150
155 160 Gln Lys Val Thr Gln Asn Phe Asn Arg Val Thr
Ala Gly Ser Leu Cys 165 170
175 Glu Thr Ser Ala Glu Phe Ile Ile Glu Asp Phe Glu Glu Cys Asn Ser
180 185 190 Asn Gly
Ser Asn Cys Gln Pro Val Pro Phe Ala Ser Phe Ser Pro Ala 195
200 205 Ile Thr Phe Ser Ser Ala Thr
Ala Thr Arg Ser Gly Arg Ser Val Ser 210 215
220 Leu Ser Gly Ala Glu Ile Thr Glu Val Ile Val Asn
Asn Gln Asp Leu 225 230 235
240 Thr Arg Cys Ser Val Ser Gly Ser Ser Thr Leu Thr Cys Ser Tyr Val
245 250 255
33236PRTTrichoderma reesei 33Met Asp Ala Ile Arg Ala Arg Ser Ala Ala Arg
Arg Ser Asn Arg Phe 1 5 10
15 Gln Ala Gly Ser Ser Lys Asn Val Asn Gly Thr Ala Asp Val Glu Ser
20 25 30 Thr Asn
Trp Ala Gly Ala Ala Ile Thr Thr Ser Gly Val Thr Glu Val 35
40 45 Ser Gly Thr Phe Thr Val Pro
Arg Pro Ser Val Pro Ala Gly Gly Ser 50 55
60 Ser Arg Glu Glu Tyr Cys Gly Ala Ala Trp Val Gly
Ile Asp Gly Tyr 65 70 75
80 Ser Asp Ala Asp Leu Ile Gln Thr Gly Val Leu Trp Cys Val Glu Asp
85 90 95 Gly Glu Tyr
Leu Tyr Glu Ala Trp Tyr Glu Tyr Leu Pro Ala Ala Leu 100
105 110 Val Glu Tyr Ser Gly Ile Ser Val
Thr Ala Gly Ser Val Val Thr Val 115 120
125 Thr Ala Thr Lys Thr Gly Thr Asn Ser Gly Val Thr Thr
Leu Thr Ser 130 135 140
Gly Gly Lys Thr Val Ser His Thr Phe Ser Arg Gln Asn Ser Pro Leu 145
150 155 160 Pro Gly Thr Ser
Ala Glu Trp Ile Val Glu Asp Phe Thr Ser Gly Ser 165
170 175 Ser Leu Val Pro Phe Ala Asp Phe Gly
Ser Val Thr Phe Thr Gly Ala 180 185
190 Thr Ala Val Val Asn Gly Ala Thr Val Thr Ala Gly Gly Asp
Ser Pro 195 200 205
Val Ile Ile Asp Leu Glu Asp Ser Arg Gly Asp Ile Leu Thr Ser Thr 210
215 220 Thr Val Ser Gly Ser
Thr Val Thr Val Glu Tyr Glu 225 230 235
34612PRTTrichoderma reesei 34Met Ala Lys Leu Ser Thr Leu Arg Leu Ala
Ser Leu Leu Ser Leu Val 1 5 10
15 Ser Val Gln Val Ser Ala Ser Val His Leu Leu Glu Ser Leu Glu
Lys 20 25 30 Leu
Pro His Gly Trp Lys Ala Ala Glu Thr Pro Ser Pro Ser Ser Gln 35
40 45 Ile Val Leu Gln Val Ala
Leu Thr Gln Gln Asn Ile Asp Gln Leu Glu 50 55
60 Ser Arg Leu Ala Ala Val Ser Thr Pro Thr Ser
Ser Thr Tyr Gly Lys 65 70 75
80 Tyr Leu Asp Val Asp Glu Ile Asn Ser Ile Phe Ala Pro Ser Asp Ala
85 90 95 Ser Ser
Ser Ala Val Glu Ser Trp Leu Gln Ser His Gly Val Thr Ser 100
105 110 Tyr Thr Lys Gln Gly Ser Ser
Ile Trp Phe Gln Thr Asn Ile Ser Thr 115 120
125 Ala Asn Ala Met Leu Ser Thr Asn Phe His Thr Tyr
Ser Asp Leu Thr 130 135 140
Gly Ala Lys Lys Val Arg Thr Leu Lys Tyr Ser Ile Pro Glu Ser Leu 145
150 155 160 Ile Gly His
Val Asp Leu Ile Ser Pro Thr Thr Tyr Phe Gly Thr Thr 165
170 175 Lys Ala Met Arg Lys Leu Lys Ser
Ser Gly Val Ser Pro Ala Ala Asp 180 185
190 Ala Leu Ala Ala Arg Gln Glu Pro Ser Ser Cys Lys Gly
Thr Leu Val 195 200 205
Phe Glu Gly Glu Thr Phe Asn Val Phe Gln Pro Asp Cys Leu Arg Thr 210
215 220 Glu Tyr Ser Val
Asp Gly Tyr Thr Pro Ser Val Lys Ser Gly Ser Arg 225 230
235 240 Ile Gly Phe Gly Ser Phe Leu Asn Glu
Ser Ala Ser Phe Ala Asp Gln 245 250
255 Ala Leu Phe Glu Lys His Phe Asn Ile Pro Ser Gln Asn Phe
Ser Val 260 265 270
Val Leu Ile Asn Gly Gly Thr Asp Leu Pro Gln Pro Pro Ser Asp Ala
275 280 285 Asn Asp Gly Glu
Ala Asn Leu Asp Ala Gln Thr Ile Leu Thr Ile Ala 290
295 300 His Pro Leu Pro Ile Thr Glu Phe
Ile Thr Ala Gly Ser Pro Pro Tyr 305 310
315 320 Phe Pro Asp Pro Val Glu Pro Ala Gly Thr Pro Asn
Glu Asn Glu Pro 325 330
335 Tyr Leu Gln Tyr Tyr Glu Phe Leu Leu Ser Lys Ser Asn Ala Glu Ile
340 345 350 Pro Gln Val
Ile Thr Asn Ser Tyr Gly Asp Glu Glu Gln Thr Val Pro 355
360 365 Arg Ser Tyr Ala Val Arg Val Cys
Asn Leu Ile Gly Leu Leu Gly Leu 370 375
380 Arg Gly Ile Ser Val Leu His Ser Ser Gly Asp Glu Gly
Val Gly Ala 385 390 395
400 Ser Cys Val Ala Thr Asn Ser Thr Thr Pro Gln Phe Asn Pro Ile Phe
405 410 415 Pro Ala Thr Cys
Pro Tyr Val Thr Ser Val Gly Gly Thr Val Ser Phe 420
425 430 Asn Pro Glu Val Ala Trp Ala Gly Ser
Ser Gly Gly Phe Ser Tyr Tyr 435 440
445 Phe Ser Arg Pro Trp Tyr Gln Gln Glu Ala Val Gly Thr Tyr
Leu Glu 450 455 460
Lys Tyr Val Ser Ala Glu Thr Lys Lys Tyr Tyr Gly Pro Tyr Val Asp 465
470 475 480 Phe Ser Gly Arg Gly
Phe Pro Asp Val Ala Ala His Ser Val Ser Pro 485
490 495 Asp Tyr Pro Val Phe Gln Gly Gly Glu Leu
Thr Pro Ser Gly Gly Thr 500 505
510 Ser Ala Ala Ser Pro Val Val Ala Ala Ile Val Ala Leu Leu Asn
Asp 515 520 525 Ala
Arg Leu Arg Glu Gly Lys Pro Thr Leu Gly Phe Leu Asn Pro Leu 530
535 540 Ile Tyr Leu His Ala Ser
Lys Gly Phe Thr Asp Ile Thr Ser Gly Gln 545 550
555 560 Ser Glu Gly Cys Asn Gly Asn Asn Thr Gln Thr
Gly Ser Pro Leu Pro 565 570
575 Gly Ala Gly Phe Ile Ala Gly Ala His Trp Asn Ala Thr Lys Gly Trp
580 585 590 Asp Pro
Thr Thr Gly Phe Gly Val Pro Asn Leu Lys Lys Leu Leu Ala 595
600 605 Leu Val Arg Phe 610
35477PRTTrichoderma reesei 35Met Arg Phe Val Gln Tyr Val Ser Leu Ala
Gly Leu Phe Ala Ala Ala 1 5 10
15 Thr Val Ser Ala Gly Val Val Thr Val Pro Phe Glu Lys Arg Asn
Leu 20 25 30 Asn
Pro Asp Phe Ala Pro Ser Leu Leu Arg Arg Asp Gly Ser Val Ser 35
40 45 Leu Asp Ala Ile Asn Asn
Leu Thr Gly Gly Gly Tyr Tyr Ala Gln Phe 50 55
60 Ser Val Gly Thr Pro Pro Gln Lys Leu Ser Phe
Leu Leu Asp Thr Gly 65 70 75
80 Ser Ser Asp Thr Trp Val Asn Ser Val Thr Ala Asp Leu Cys Thr Asp
85 90 95 Glu Phe
Thr Gln Gln Thr Val Gly Glu Tyr Cys Phe Arg Gln Phe Asn 100
105 110 Pro Arg Arg Ser Ser Ser Tyr
Lys Ala Ser Thr Glu Val Phe Asp Ile 115 120
125 Thr Tyr Leu Asp Gly Arg Arg Ile Arg Gly Asn Tyr
Phe Thr Asp Thr 130 135 140
Val Thr Ile Asn Gln Ala Asn Ile Thr Gly Gln Lys Ile Gly Leu Ala 145
150 155 160 Leu Gln Ser
Val Arg Gly Thr Gly Ile Leu Gly Leu Gly Phe Arg Glu 165
170 175 Asn Glu Ala Ala Asp Thr Lys Tyr
Pro Thr Val Ile Asp Asn Leu Val 180 185
190 Ser Gln Lys Val Ile Pro Val Pro Ala Phe Ser Leu Tyr
Leu Asn Asp 195 200 205
Leu Gln Thr Ser Gln Gly Ile Leu Leu Phe Gly Gly Val Asp Thr Asp 210
215 220 Lys Phe His Gly
Gly Leu Ala Thr Leu Pro Leu Gln Ser Leu Pro Pro 225 230
235 240 Ser Ile Ala Glu Thr Gln Asp Ile Val
Met Tyr Ser Val Asn Leu Asp 245 250
255 Gly Phe Ser Ala Ser Asp Val Asp Thr Pro Asp Val Ser Ala
Lys Ala 260 265 270
Val Leu Asp Ser Gly Ser Thr Ile Thr Leu Leu Pro Asp Ala Val Val
275 280 285 Gln Glu Leu Phe
Asp Glu Tyr Asp Val Leu Asn Ile Gln Gly Leu Pro 290
295 300 Val Pro Phe Ile Asp Cys Ala Lys
Ala Asn Ile Lys Asp Ala Thr Phe 305 310
315 320 Asn Phe Lys Phe Asp Gly Lys Thr Ile Lys Val Pro
Ile Asp Glu Met 325 330
335 Val Leu Asn Asn Leu Ala Ala Ala Ser Asp Glu Ile Met Ser Asp Pro
340 345 350 Ser Leu Ser
Lys Phe Phe Lys Gly Trp Ser Gly Val Cys Thr Phe Gly 355
360 365 Met Gly Ser Thr Lys Thr Phe Gly
Ile Gln Ser Asp Glu Phe Val Leu 370 375
380 Leu Gly Asp Thr Phe Leu Arg Ser Ala Tyr Val Val Tyr
Asp Leu Gln 385 390 395
400 Asn Lys Gln Ile Gly Ile Ala Gln Ala Thr Leu Asn Ser Thr Ser Ser
405 410 415 Thr Ile Val Glu
Phe Lys Ala Gly Ser Lys Thr Ile Pro Gly Pro Ala 420
425 430 Ser Thr Gly Asp Asp Ser Asp Asp Ser
Ser Asp Asp Ser Asp Glu Asp 435 440
445 Ser Ala Gly Ala Ala Leu His Pro Thr Phe Ser Ile Ala Leu
Ala Gly 450 455 460
Thr Leu Phe Thr Ala Val Ser Met Met Met Ser Val Leu 465
470 475 361263DNATrichoderma reesei 36atggcgtcac
tcatcaaaac tgccgtggac attgccaacg gccgccatgc gctgtccaga 60tatgtcatct
ttgggctctg gcttgcggat gcggtgctgt gcgggctgat tatctggaaa 120gtgccttata
cggaaatcga ctgggtcgcc tacatggagc aagtcaccca gttcgtccac 180ggagagcgag
actaccccaa gatggagggc ggcacagggc ccctggtgta tcccgcggcc 240catgtgtaca
tctacacagg gctctactac ctgacgaaca agggcaccga catcctgctg 300gcgcagcagc
tctttgccgt gctctacatg gctactctgg cggtcgtcat gacatgctac 360tccaaggcca
aggtcccgcc gtacatcttc ccgcttctca tcctctccaa aagacttcac 420agcgtcttcg
tcctgagatg cttcaacgac tgcttcgccg ccttcttcct ctggctctgc 480atcttcttct
tccagaggcg agagtggacc atcggagctc tcgcatacag catcggcctg 540ggcgtcaaaa
tgtcgctgct actggttctc cccgccgtgg tcatcgtcct ctacctcggc 600cgcggcttca
agggcgccct gcggctgctc tggctcatgg tgcaggtcca gctcctcctc 660gccataccct
tcatcacgac aaattggcgc ggctacctcg gccgtgcatt cgagctctcg 720aggcagttca
agtttgaatg gacagtcaat tggcgcatgc tgggcgagga tctgttcctc 780agccggggct
tctctatcac gctactggca tttcacgcca tcttcctcct cgcctttatc 840ctcggccggt
ggctgaagat tagggaacgg accgtactcg ggatgatccc ctatgtcatc 900cgattcagat
cgccctttac cgagcaggaa gagcgcgcca tctccaaccg cgtcgtcacg 960cccggctatg
tcatgtccac catcttgtcg gccaacgtgg tgggactgct gtttgcccgg 1020tctctgcact
accagttcta tgcatatctg gcgtgggcga ccccctatct cctgtggacg 1080gcctgcccca
atcttttggt ggtggccccc ctctgggcgg cgcaagaatg ggcctggaac 1140gtcttcccca
gcacgcctct tagctcgagc gtcgtggtga gcgtgctggc cgtgacggtg 1200gccatggcgt
ttgcaggttc aaatccgcag ccacgtgaaa catcgaagcc gaagcagcac 1260taa
126337420PRTTrichoderma reesei 37Met Ala Ser Leu Ile Lys Thr Ala Val Asp
Ile Ala Asn Gly Arg His 1 5 10
15 Ala Leu Ser Arg Tyr Val Ile Phe Gly Leu Trp Leu Ala Asp Ala
Val 20 25 30 Leu
Cys Gly Leu Ile Ile Trp Lys Val Pro Tyr Thr Glu Ile Asp Trp 35
40 45 Val Ala Tyr Met Glu
Gln Val Thr Gln Phe Val His Gly Glu Arg Asp 50 55
60 Tyr Pro Lys Met Glu Gly Gly Thr Gly Pro
Leu Val Tyr Pro Ala Ala 65 70 75
80 His Val Tyr Ile Tyr Thr Gly Leu Tyr Tyr Leu Thr Asn Lys Gly
Thr 85 90 95 Asp
Ile Leu Leu Ala Gln Gln Leu Phe Ala Val Leu Tyr Met Ala Thr
100 105 110 Leu Ala Val Val Met
Thr Cys Tyr Ser Lys Ala Lys Val Pro Pro Tyr 115
120 125 Ile Phe Pro Leu Leu Ile Leu Ser Lys
Arg Leu His Ser Val Phe Val 130 135
140 Leu Arg Cys Phe Asn Asp Cys Phe Ala Ala Phe Phe Leu
Trp Leu Cys 145 150 155
160 Ile Phe Phe Phe Gln Arg Arg Glu Trp Thr Ile Gly Ala Leu Ala Tyr
165 170 175 Ser Ile Gly Leu
Gly Val Lys Met Ser Leu Leu Leu Val Leu Pro Ala 180
185 190 Val Val Ile Val Leu Tyr Leu Gly Arg
Gly Phe Lys Gly Ala Leu Arg 195 200
205 Leu Leu Trp Leu Met Val Gln Val Gln Leu Leu Leu Ala Ile
Pro Phe 210 215 220
Ile Thr Thr Asn Trp Arg Gly Tyr Leu Gly Arg Ala Phe Glu Leu Ser 225
230 235 240 Arg Gln Phe Lys Phe
Glu Trp Thr Val Asn Trp Arg Met Leu Gly Glu 245
250 255 Asp Leu Phe Leu Ser Arg Gly Phe Ser Ile
Thr Leu Leu Ala Phe His 260 265
270 Ala Ile Phe Leu Leu Ala Phe Ile Leu Gly Arg Trp Leu Lys Ile
Arg 275 280 285 Glu
Arg Thr Val Leu Gly Met Ile Pro Tyr Val Ile Arg Phe Arg Ser 290
295 300 Pro Phe Thr Glu Gln Glu
Glu Arg Ala Ile Ser Asn Arg Val Val Thr 305 310
315 320 Pro Gly Tyr Val Met Ser Thr Ile Leu Ser Ala
Asn Val Val Gly Leu 325 330
335 Leu Phe Ala Arg Ser Leu His Tyr Gln Phe Tyr Ala Tyr Leu Ala Trp
340 345 350 Ala Thr
Pro Tyr Leu Leu Trp Thr Ala Cys Pro Asn Leu Leu Val Val 355
360 365 Ala Pro Leu Trp Ala Ala Gln
Glu Trp Ala Trp Asn Val Phe Pro Ser 370 375
380 Thr Pro Leu Ser Ser Ser Val Val Val Ser Val Leu
Ala Val Thr Val 385 390 395
400 Ala Met Ala Phe Ala Gly Ser Asn Pro Gln Pro Arg Glu Thr Ser Lys
405 410 415 Pro Lys Gln
His 420 38445PRTHomo sapiens 38Met Leu Lys Lys Gln Ser Ala
Gly Leu Val Leu Trp Gly Ala Ile Leu 1 5
10 15 Phe Val Ala Trp Asn Ala Leu Leu Leu Leu Phe
Phe Trp Thr Arg Pro 20 25
30 Ala Pro Gly Arg Pro Pro Ser Val Ser Ala Leu Asp Gly Asp Pro
Ala 35 40 45 Ser
Leu Thr Arg Glu Val Ile Arg Leu Ala Gln Asp Ala Glu Val Glu 50
55 60 Leu Glu Arg Gln Arg Gly
Leu Leu Gln Gln Ile Gly Asp Ala Leu Ser 65 70
75 80 Ser Gln Arg Gly Arg Val Pro Thr Ala Ala Pro
Pro Ala Gln Pro Arg 85 90
95 Val Pro Val Thr Pro Ala Pro Ala Val Ile Pro Ile Leu Val Ile Ala
100 105 110 Cys Asp
Arg Ser Thr Val Arg Arg Cys Leu Asp Lys Leu Leu His Tyr 115
120 125 Arg Pro Ser Ala Glu Leu Phe
Pro Ile Ile Val Ser Gln Asp Cys Gly 130 135
140 His Glu Glu Thr Ala Gln Ala Ile Ala Ser Tyr Gly
Ser Ala Val Thr 145 150 155
160 His Ile Arg Gln Pro Asp Leu Ser Ser Ile Ala Val Pro Pro Asp His
165 170 175 Arg Lys Phe
Gln Gly Tyr Tyr Lys Ile Ala Arg His Tyr Arg Trp Ala 180
185 190 Leu Gly Gln Val Phe Arg Gln Phe
Arg Phe Pro Ala Ala Val Val Val 195 200
205 Glu Asp Asp Leu Glu Val Ala Pro Asp Phe Phe Glu Tyr
Phe Arg Ala 210 215 220
Thr Tyr Pro Leu Leu Lys Ala Asp Pro Ser Leu Trp Cys Val Ser Ala 225
230 235 240 Trp Asn Asp Asn
Gly Lys Glu Gln Met Val Asp Ala Ser Arg Pro Glu 245
250 255 Leu Leu Tyr Arg Thr Asp Phe Phe Pro
Gly Leu Gly Trp Leu Leu Leu 260 265
270 Ala Glu Leu Trp Ala Glu Leu Glu Pro Lys Trp Pro Lys Ala
Phe Trp 275 280 285
Asp Asp Trp Met Arg Arg Pro Glu Gln Arg Gln Gly Arg Ala Cys Ile 290
295 300 Arg Pro Glu Ile Ser
Arg Thr Met Thr Phe Gly Arg Lys Gly Val Ser 305 310
315 320 His Gly Gln Phe Phe Asp Gln His Leu Lys
Phe Ile Lys Leu Asn Gln 325 330
335 Gln Phe Val His Phe Thr Gln Leu Asp Leu Ser Tyr Leu Gln Arg
Glu 340 345 350 Ala
Tyr Asp Arg Asp Phe Leu Ala Arg Val Tyr Gly Ala Pro Gln Leu 355
360 365 Gln Val Glu Lys Val Arg
Thr Asn Asp Arg Lys Glu Leu Gly Glu Val 370 375
380 Arg Val Gln Tyr Thr Gly Arg Asp Ser Phe Lys
Ala Phe Ala Lys Ala 385 390 395
400 Leu Gly Val Met Asp Asp Leu Lys Ser Gly Val Pro Arg Ala Gly Tyr
405 410 415 Arg Gly
Ile Val Thr Phe Gln Phe Arg Gly Arg Arg Val His Leu Ala 420
425 430 Pro Pro Leu Thr Trp Glu Gly
Tyr Asp Pro Ser Trp Asn 435 440
445 39447PRTHomo sapiens 39Met Arg Phe Arg Ile Tyr Lys Arg Lys Val Leu
Ile Leu Thr Leu Val 1 5 10
15 Val Ala Ala Cys Gly Phe Val Leu Trp Ser Ser Asn Gly Arg Gln Arg
20 25 30 Lys Asn
Glu Ala Leu Ala Pro Pro Leu Leu Asp Ala Glu Pro Ala Arg 35
40 45 Gly Ala Gly Gly Arg Gly Gly
Asp His Pro Ser Val Ala Val Gly Ile 50 55
60 Arg Arg Val Ser Asn Val Ser Ala Ala Ser Leu Val
Pro Ala Val Pro 65 70 75
80 Gln Pro Glu Ala Asp Asn Leu Thr Leu Arg Tyr Arg Ser Leu Val Tyr
85 90 95 Gln Leu Asn
Phe Asp Gln Thr Leu Arg Asn Val Asp Lys Ala Gly Thr 100
105 110 Trp Ala Pro Arg Glu Leu Val Leu
Val Val Gln Val His Asn Arg Pro 115 120
125 Glu Tyr Leu Arg Leu Leu Leu Asp Ser Leu Arg Lys Ala
Gln Gly Ile 130 135 140
Asp Asn Val Leu Val Ile Phe Ser His Asp Phe Trp Ser Thr Glu Ile 145
150 155 160 Asn Gln Leu Ile
Ala Gly Val Asn Phe Cys Pro Val Leu Gln Val Phe 165
170 175 Phe Pro Phe Ser Ile Gln Leu Tyr Pro
Asn Glu Phe Pro Gly Ser Asp 180 185
190 Pro Arg Asp Cys Pro Arg Asp Leu Pro Lys Asn Ala Ala Leu
Lys Leu 195 200 205
Gly Cys Ile Asn Ala Glu Tyr Pro Asp Ser Phe Gly His Tyr Arg Glu 210
215 220 Ala Lys Phe Ser Gln
Thr Lys His His Trp Trp Trp Lys Leu His Phe 225 230
235 240 Val Trp Glu Arg Val Lys Ile Leu Arg Asp
Tyr Ala Gly Leu Ile Leu 245 250
255 Phe Leu Glu Glu Asp His Tyr Leu Ala Pro Asp Phe Tyr His Val
Phe 260 265 270 Lys
Lys Met Trp Lys Leu Lys Gln Gln Glu Cys Pro Glu Cys Asp Val 275
280 285 Leu Ser Leu Gly Thr Tyr
Ser Ala Ser Arg Ser Phe Tyr Gly Met Ala 290 295
300 Asp Lys Val Asp Val Lys Thr Trp Lys Ser Thr
Glu His Asn Met Gly 305 310 315
320 Leu Ala Leu Thr Arg Asn Ala Tyr Gln Lys Leu Ile Glu Cys Thr Asp
325 330 335 Thr Phe
Cys Thr Tyr Asp Asp Tyr Asn Trp Asp Trp Thr Leu Gln Tyr 340
345 350 Leu Thr Val Ser Cys Leu Pro
Lys Phe Trp Lys Val Leu Val Pro Gln 355 360
365 Ile Pro Arg Ile Phe His Ala Gly Asp Cys Gly Met
His His Lys Lys 370 375 380
Thr Cys Arg Pro Ser Thr Gln Ser Ala Gln Ile Glu Ser Leu Leu Asn 385
390 395 400 Asn Asn Lys
Gln Tyr Met Phe Pro Glu Thr Leu Thr Ile Ser Glu Lys 405
410 415 Phe Thr Val Val Ala Ile Ser Pro
Pro Arg Lys Asn Gly Gly Trp Gly 420 425
430 Asp Ile Arg Asp His Glu Leu Cys Lys Ser Tyr Arg Arg
Leu Gln 435 440 445
4085PRTTrichoderma reesei 40Met Ala Ser Thr Asn Ala Arg Tyr Val Arg Tyr
Leu Leu Ile Ala Phe 1 5 10
15 Phe Thr Ile Leu Val Phe Tyr Phe Val Ser Asn Ser Lys Tyr Glu Gly
20 25 30 Val Asp
Leu Asn Lys Gly Thr Phe Thr Ala Pro Asp Ser Thr Lys Thr 35
40 45 Thr Pro Lys Pro Pro Ala Thr
Gly Asp Ala Lys Asp Phe Pro Leu Ala 50 55
60 Leu Thr Pro Asn Asp Pro Gly Phe Asn Asp Leu Val
Gly Ile Ala Pro 65 70 75
80 Gly Pro Arg Met Asn 85 41255DNATrichoderma reesei
41atggcgtcaa caaatgcgcg ctatgtgcgc tatctactaa tcgccttctt cacaatcctc
60gtcttctact ttgtctccaa ttcaaagtat gagggcgtcg atctcaacaa gggcaccttc
120acagctccgg attcgaccaa gacgacacca aagccgccag ccactggcga tgccaaagac
180tttcctctgg ccctgacgcc gaacgatcca ggcttcaacg acctcgtcgg catcgctccc
240ggccctcgaa tgaac
2554258PRTHomo sapiens 42Met Arg Phe Arg Ile Tyr Lys Arg Lys Val Leu Ile
Leu Thr Leu Val 1 5 10
15 Val Ala Ala Cys Gly Phe Val Leu Trp Ser Ser Asn Gly Arg Gln Arg
20 25 30 Lys Asn Glu
Ala Leu Ala Pro Pro Leu Leu Asp Ala Glu Pro Ala Arg 35
40 45 Gly Ala Gly Gly Arg Gly Gly Asp
His Pro 50 55 4351PRTTrichoderma reesei
43Met Ala Ser Thr Asn Ala Arg Tyr Val Arg Tyr Leu Leu Ile Ala Phe 1
5 10 15 Phe Thr Ile Leu
Val Phe Tyr Phe Val Ser Asn Ser Lys Tyr Glu Gly 20
25 30 Val Asp Leu Asn Lys Gly Thr Phe Thr
Ala Pro Asp Ser Thr Lys Thr 35 40
45 Thr Pro Lys 50 4452PRTTrichoderma reesei 44Met
Ala Ile Ala Arg Pro Val Arg Ala Leu Gly Gly Leu Ala Ala Ile 1
5 10 15 Leu Trp Cys Phe Phe Leu
Tyr Gln Leu Leu Arg Pro Ser Ser Ser Tyr 20
25 30 Asn Ser Pro Gly Asp Arg Tyr Ile Asn Phe
Glu Arg Asp Pro Asn Leu 35 40
45 Asp Pro Thr Gly 50 4533PRTTrichoderma reesei
45Met Leu Asn Pro Arg Arg Ala Leu Ile Ala Ala Ala Phe Ile Leu Thr 1
5 10 15 Val Phe Phe Leu
Ile Ser Arg Ser His Asn Ser Glu Ser Ala Ser Thr 20
25 30 Ser 4684PRTTrichoderma reesei 46Met
Met Pro Arg His His Ser Ser Gly Phe Ser Asn Gly Tyr Pro Arg 1
5 10 15 Ala Asp Thr Phe Glu Ile
Ser Pro His Arg Phe Gln Pro Arg Ala Thr 20
25 30 Leu Pro Pro His Arg Lys Arg Lys Arg Thr
Ala Ile Arg Val Gly Ile 35 40
45 Ala Val Val Val Ile Leu Val Leu Val Leu Trp Phe Gly Gln
Pro Arg 50 55 60
Ser Val Ala Ser Leu Ile Ser Leu Gly Ile Leu Ser Gly Tyr Asp Asp 65
70 75 80 Leu Lys Leu Glu
4755PRTTrichoderma reesei 47Met Leu Leu Pro Lys Gly Gly Leu Asp Trp Arg
Ser Ala Arg Ala Gln 1 5 10
15 Ile Pro Pro Thr Arg Ala Leu Trp Asn Ala Val Thr Arg Thr Arg Phe
20 25 30 Ile Leu
Leu Val Gly Ile Thr Gly Leu Ile Leu Leu Leu Trp Arg Gly 35
40 45 Val Ser Thr Ser Ala Ser Glu
50 55 4869DNAArtificial SequencePrimer 48cgattaagtt
gggtaacgcc agggttttcc cagtcacgac ggtttaaacg ctgcagggcg 60tacagaact
694969DNAArtificial SequencePrimer 49atctctcaaa ggaagaatcc cttcagggtt
gcgtttccag tgcggccgcg gctctaaaat 60gcttcacag
695068DNAArtificial SequencePrimer
50cggttctcat ctgggcttgc tcggtcctgg cgtagatcta gcggccgcac gatgatgatg
60acagccag
685169DNAArtificial SequencePrimer 51gtggaattgt gagcggataa caatttcaca
caggaaacag cgtttaaacc gtccagctcc 60cgcagcgcc
695284DNAArtificial SequencePrimer
52atcgctaact gctttctctt ctgtgaagca ttttagagcc gcggccgcgg ccggccgcga
60tcgcctagat ctacgccagg accg
845348DNAArtificial SequencePrimer 53cggtcctggc gtagatctag ggcgcgccac
tggaaacgca accctgaa 485448DNAArtificial SequencePrimer
54ttcagggttg cgtttccagt ggcgcgccct agatctacgc caggaccg
485568DNAArtificial SequencePrimer 55agcatcatga ccgccccctt ctggctgtca
tcatcatcgt gcggccgcga ttattgcaca 60agcagcga
685620DNAArtificial SequencePrimer
56tatggcttta gatggggaca
205720DNAArtificial SequencePrimer 57tgcgtcgccg tctcgctcct
205820DNAArtificial SequencePrimer
58ttaggcgacc tctttttcca
205918DNAArtificial SequencePrimer 59cctgtatcgt cctgttcc
186020DNAArtificial SequencePrimer
60gcgcctgtcg agtcggcatt
206120DNAArtificial SequencePrimer 61caccggccat gctcttgcca
206218DNAArtificial SequencePrimer
62caaggtgccc tatgtcgc
186318DNAArtificial SequencePrimer 63gatcgggtca ggacggaa
186418DNAArtificial SequencePrimer
64agcctgtctg agggacgg
186518DNAArtificial SequencePrimer 65caaggtcgag attcggca
186617DNAArtificial SequencePrimer
66cagaaggggg cggtcat
176717DNAArtificial SequencePrimer 67gtcccagctc ccgctct
176820DNAArtificial SequencePrimer
68gcgcctgtcg agtcggcatt
206920DNAArtificial SequencePrimer 69caccggccat gctcttgcca
207069DNAArtificial SequencePrimer
70cgattaagtt gggtaacgcc agggttttcc cagtcacgac ggtttaaacg tttcaggtac
60caacacctg
697169DNAArtificial SequencePrimer 71atctctcaaa ggaagaatcc cttcagggtt
gcgtttccag tgcggccgcg gcgaagagtc 60tggcgggga
697268DNAArtificial SequencePrimer
72cggttctcat ctgggcttgc tcggtcctgg cgtagatcta gcggccgcaa gaggatgggg
60gtaaagct
687369DNAArtificial SequencePrimer 73gtggaattgt gagcggataa caatttcaca
caggaaacag cgtttaaacg aggaggactc 60gtgagttat
697484DNAArtificial SequencePrimer
74gcgcccttcc gcctcgacaa tccccgccag actcttcgcc gcggccgcgg ccggccgcga
60tcgcctagat ctacgccagg accg
847548DNAArtificial SequencePrimer 75cggtcctggc gtagatctag ggcgcgccac
tggaaacgca accctgaa 487648DNAArtificial SequencePrimer
76ttcagggttg cgtttccagt ggcgcgccct agatctacgc caggaccg
487768DNAArtificial SequencePrimer 77gagctggcca gaaaagacca agctttaccc
ccatcctctt gcggccgcga ttattgcaca 60agcagcga
687820DNAArtificial SequencePrimer
78acgagttgtt tcgtgtaccg
207921DNAArtificial SequencePrimer 79ctttccattc atcagggatg g
218020DNAArtificial SequencePrimer
80ggagactcag tgaagagagg
208118DNAArtificial SequencePrimer 81atgttgcagt tgcgaaag
188220DNAArtificial SequencePrimer
82ccctcgtcgc agaaaagatg
208320DNAArtificial SequencePrimer 83agcctccttg ggaacctcag
208420DNAArtificial SequencePrimer
84cttagtgcgg ctggagggcg
208520DNAArtificial SequencePrimer 85ggccggttcg tgcaactgga
208620DNAArtificial SequencePrimer
86ggccgcaaga ggatgggggt
208721DNAArtificial SequencePrimer 87tcgggccagc tgaagcacaa c
218820DNAArtificial SequencePrimer
88ttgaggaacg gctgcctgcg
208920DNAArtificial SequencePrimer 89cgatggctcc gtcatccgcc
209020DNAArtificial SequencePrimer
90acgagttgtt tcgtgtaccg
209120DNAArtificial SequencePrimer 91tgcgtcgccg tctcgctcct
209220DNAArtificial SequencePrimer
92ttaggcgacc tctttttcca
209318DNAArtificial SequencePrimer 93atgttgcagt tgcgaaag
189420DNAArtificial SequencePrimer
94ccctcgtcgc agaaaagatg
209520DNAArtificial SequencePrimer 95agcctccttg ggaacctcag
209620DNAArtificial SequencePrimer
96cttagtgcgg ctggagggcg
209720DNAArtificial SequencePrimer 97ggccggttcg tgcaactgga
209820DNAArtificial SequencePrimer
98ggccgcaaga ggatgggggt
209921DNAArtificial SequencePrimer 99tcgggccagc tgaagcacaa c
2110020DNAArtificial SequencePrimer
100ccctcgtcgc agaaaagatg
2010120DNAArtificial SequencePrimer 101agcctccttg ggaacctcag
2010269DNAArtificial SequencePrimer
102cgattaagtt gggtaacgcc agggttttcc cagtcacgac ggtttaaacg tgtttaaatt
60tgatgaggc
6910369DNAArtificial SequencePrimer 103atctctcaaa ggaagaatcc cttcagggtt
gcgtttccag tgcggccgcg gtctcagaga 60cagccttct
6910468DNAArtificial SequencePrimer
104cggttctcat ctgggcttgc tcggtcctgg cgtagatcta gcggccgcac tcggcttctt
60tgtccgag
6810569DNAArtificial SequencePrimer 105gtggaattgt gagcggataa caatttcaca
caggaaacag cgtttaaact cctcgtcggc 60aacaaggcc
6910684DNAArtificial SequencePrimer
106gcagatctgg gggaggaatc agaaggctgt ctctgagacc gcggccgcgg ccggccgcga
60tcgcctagat ctacgccagg accg
8410748DNAArtificial SequencePrimer 107cggtcctggc gtagatctag ggcgcgccac
tggaaacgca accctgaa 4810848DNAArtificial SequencePrimer
108ttcagggttg cgtttccagt ggcgcgccct agatctacgc caggaccg
4810968DNAArtificial SequencePrimer 109aaagtgggcg agctgagata ctcggacaaa
gaagccgagt gcggccgcga ttattgcaca 60agcagcga
6811018DNAArtificial SequencePrimer
110acgggagatc tcggaaaa
1811121DNAArtificial SequencePrimer 111ctttccattc atcagggatg g
2111220DNAArtificial SequencePrimer
112ggagactcag tgaagagagg
2011318DNAArtificial SequencePrimer 113atgaagctca gcctgtgg
1811418DNAArtificial SequencePrimer
114ggggacggct tgaggaag
1811520DNAArtificial SequencePrimer 115ctgcttgctg cttccagtca
2011620DNAArtificial SequencePrimer
116tggcagatgc cgaaaggcgg
2011720DNAArtificial SequencePrimer 117tggcaaccag ctgtggctcc
2011820DNAArtificial SequencePrimer
118cggccgcact cggcttcttt
2011920DNAArtificial SequencePrimer 119gagtgggcta ggcgcaacgg
2012020DNAArtificial SequencePrimer
120ggatcggcca ctgccaccac
2012120DNAArtificial SequencePrimer 121gcccacttct ctgcgcgtgt
2012218DNAArtificial SequencePrimer
122acgggagatc tcggaaaa
1812320DNAArtificial SequencePrimer 123ccatgagctt gaacaggtaa
2012420DNAArtificial SequencePrimer
124ttaggcgacc tctttttcca
2012518DNAArtificial SequencePrimer 125atgaagctca gcctgtgg
1812620DNAArtificial SequencePrimer
126ggatcggcca ctgccaccac
2012720DNAArtificial SequencePrimer 127gcccacttct ctgcgcgtgt
2012820DNAArtificial SequencePrimer
128tggcagatgc cgaaaggcgg
2012920DNAArtificial SequencePrimer 129tggcaaccag ctgtggctcc
2013020DNAArtificial SequencePrimer
130cggccgcact cggcttcttt
2013120DNAArtificial SequencePrimer 131gagtgggcta ggcgcaacgg
2013217DNAArtificial SequencePrimer
132ctctgcgcgt gttgtgg
1713317DNAArtificial SequencePrimer 133taagggtgcg gattcgg
17134488PRTTrichoderma reesei 134Met
Arg Ala Ser Pro Leu Ala Val Ala Gly Val Ala Leu Ala Ser Ala 1
5 10 15 Ala Gln Ala Gln Val Val
Gln Phe Asp Ile Glu Lys Arg His Ala Pro 20
25 30 Arg Leu Ser Arg Arg Asp Gly Thr Ile Asp
Gly Thr Leu Ser Asn Gln 35 40
45 Arg Val Gln Gly Gly Tyr Phe Ile Asn Val Gln Val Gly Ser
Pro Gly 50 55 60
Gln Asn Ile Thr Leu Gln Leu Asp Thr Gly Ser Ser Asp Val Trp Val 65
70 75 80 Pro Ser Ser Thr Ala
Ala Ile Cys Thr Gln Val Ser Glu Arg Asn Pro 85
90 95 Gly Cys Gln Phe Gly Ser Phe Asn Pro Asp
Asp Ser Asp Thr Phe Asp 100 105
110 Glu Val Gly Gln Gly Leu Phe Asp Ile Thr Tyr Val Asp Gly Ser
Ser 115 120 125 Ser
Lys Gly Asp Tyr Phe Gln Asp Asn Phe Gln Ile Asn Gly Val Thr 130
135 140 Val Lys Asn Leu Thr Met
Gly Leu Gly Leu Ser Ser Ser Ile Pro Asn 145 150
155 160 Gly Leu Ile Gly Val Gly Tyr Met Asn Asp Glu
Ala Ser Val Ser Thr 165 170
175 Thr Arg Ser Thr Tyr Pro Asn Leu Pro Ile Val Leu Gln Gln Gln Lys
180 185 190 Leu Ile
Asn Ser Val Ala Phe Ser Leu Trp Leu Asn Asp Leu Asp Ala 195
200 205 Ser Thr Gly Ser Ile Leu Phe
Gly Gly Ile Asp Thr Glu Lys Tyr His 210 215
220 Gly Asp Leu Thr Ser Ile Asp Ile Ile Ser Pro Asn
Gly Gly Lys Thr 225 230 235
240 Phe Thr Glu Phe Ala Val Asn Leu Tyr Ser Val Gln Ala Thr Ser Pro
245 250 255 Ser Gly Thr
Asp Thr Leu Ser Thr Ser Glu Asp Thr Leu Ile Ala Val 260
265 270 Leu Asp Ser Gly Thr Thr Leu Thr
Tyr Leu Pro Gln Asp Met Ala Glu 275 280
285 Glu Ala Trp Asn Glu Val Gly Ala Glu Tyr Ser Asn Glu
Leu Gly Leu 290 295 300
Ala Val Val Pro Cys Ser Val Gly Asn Thr Asn Gly Phe Phe Ser Phe 305
310 315 320 Thr Phe Ala Gly
Thr Asp Gly Pro Thr Ile Asn Val Thr Leu Ser Glu 325
330 335 Leu Val Leu Asp Leu Phe Ser Gly Gly
Pro Ala Pro Arg Phe Ser Ser 340 345
350 Gly Pro Asn Lys Gly Gln Ser Ile Cys Glu Phe Gly Ile Gln
Asn Gly 355 360 365
Thr Gly Ser Pro Phe Leu Leu Gly Asp Thr Phe Leu Arg Ser Ala Phe 370
375 380 Val Val Tyr Asp Leu
Val Asn Asn Gln Ile Ala Ile Ala Pro Thr Asn 385 390
395 400 Phe Asn Ser Thr Arg Thr Asn Val Val Ala
Phe Ala Ser Ser Gly Ala 405 410
415 Pro Ile Pro Ser Ala Thr Ala Ala Pro Asn Gln Ser Arg Thr Gly
His 420 425 430 Ser
Ser Ser Thr His Ser Gly Leu Ser Ala Ala Ser Gly Phe His Asp 435
440 445 Gly Asp Asp Glu Asn Ala
Gly Ser Leu Thr Ser Val Phe Ser Gly Pro 450 455
460 Gly Met Ala Val Val Gly Met Thr Ile Cys Tyr
Thr Leu Leu Gly Ser 465 470 475
480 Ala Ile Phe Gly Ile Gly Trp Leu 485
135761PRTTrichoderma reesei 135Met Arg Ser Thr Leu Tyr Gly Leu Ala Ala
Leu Pro Leu Ala Ala Gln 1 5 10
15 Ala Leu Glu Phe Ile Asp Asp Thr Val Ala Gln Gln Asn Gly Ile
Met 20 25 30 Arg
Tyr Thr Leu Thr Thr Thr Lys Gly Ala Thr Ser Lys His Leu His 35
40 45 Arg Arg Gln Asp Ser Ala
Asp Leu Met Ser Gln Gln Thr Gly Tyr Phe 50 55
60 Tyr Ser Ile Gln Leu Glu Ile Gly Thr Pro Pro
Gln Ala Val Ser Val 65 70 75
80 Asn Phe Asp Thr Gly Ser Ser Glu Leu Trp Val Asn Pro Val Cys Ser
85 90 95 Lys Ala
Thr Asp Pro Ala Phe Cys Lys Thr Phe Gly Gln Tyr Asn His 100
105 110 Ser Thr Thr Phe Val Asp Ala
Lys Ala Pro Gly Gly Ile Lys Tyr Gly 115 120
125 Thr Gly Phe Val Asp Phe Asn Tyr Gly Tyr Asp Tyr
Val Gln Leu Gly 130 135 140
Ser Leu Arg Ile Asn Gln Gln Val Phe Gly Val Ala Thr Asp Ser Glu 145
150 155 160 Phe Ala Ser
Val Gly Ile Leu Gly Ala Gly Pro Asp Leu Ser Gly Trp 165
170 175 Thr Ser Pro Tyr Pro Phe Val Ile
Asp Asn Leu Val Lys Gln Gly Phe 180 185
190 Ile Lys Ser Arg Ala Phe Ser Leu Asp Ile Arg Gly Leu
Asp Ser Asp 195 200 205
Arg Gly Ser Val Thr Tyr Gly Gly Ile Asp Ile Lys Lys Phe Ser Gly 210
215 220 Pro Leu Ala Lys
Lys Pro Ile Ile Pro Ala Ala Gln Ser Pro Asp Gly 225 230
235 240 Tyr Thr Arg Tyr Trp Val His Met Asp
Gly Met Ser Ile Thr Lys Glu 245 250
255 Asp Gly Ser Lys Phe Glu Ile Phe Asp Lys Pro Asn Gly Gln
Pro Val 260 265 270
Leu Leu Asp Ser Gly Tyr Thr Val Ser Thr Leu Pro Gly Pro Leu Met
275 280 285 Asp Lys Ile Leu
Glu Ala Phe Pro Ser Ala Arg Leu Glu Ser Thr Ser 290
295 300 Gly Asp Tyr Ile Val Asp Cys Asp
Ile Ile Asp Thr Pro Gly Arg Val 305 310
315 320 Asn Phe Lys Phe Gly Asn Val Val Val Asp Val Glu
Tyr Lys Asp Phe 325 330
335 Ile Trp Gln Gln Pro Asp Leu Gly Ile Cys Lys Leu Gly Val Ser Gln
340 345 350 Asp Asp Asn
Phe Pro Val Leu Gly Asp Thr Phe Leu Arg Ala Ala Tyr 355
360 365 Val Val Phe Asp Trp Asp Asn Gln
Glu Val His Ile Ala Ala Asn Glu 370 375
380 Asp Cys Gly Asp Glu Leu Ile Pro Ile Gly Ser Gly Pro
Asp Ala Ile 385 390 395
400 Pro Ala Ser Ala Ile Gly Lys Cys Ser Pro Ser Val Lys Thr Asp Thr
405 410 415 Thr Thr Ser Val
Ala Glu Thr Thr Ala Thr Ser Ala Ala Ala Ser Thr 420
425 430 Ser Glu Leu Ala Ala Thr Thr Ser Glu
Ala Ala Thr Thr Ser Ser Glu 435 440
445 Ala Ala Thr Thr Ser Ala Ala Ala Glu Thr Thr Ser Val Pro
Leu Asn 450 455 460
Thr Ala Pro Ala Thr Thr Gly Leu Leu Pro Thr Thr Ser His Arg Phe 465
470 475 480 Ser Asn Gly Thr Ala
Pro Tyr Pro Ile Pro Ser Leu Ser Ser Val Ala 485
490 495 Ala Ala Ala Gly Ser Ser Thr Val Pro Ser
Glu Ser Ser Thr Gly Ala 500 505
510 Ala Ala Ala Gly Thr Thr Ser Ala Ala Thr Gly Ser Gly Ser Gly
Ser 515 520 525 Gly
Ser Gly Asp Ala Thr Thr Ala Ser Ala Thr Tyr Thr Ser Thr Phe 530
535 540 Thr Thr Thr Asn Val Tyr
Thr Val Thr Ser Cys Pro Pro Ser Val Thr 545 550
555 560 Asn Cys Pro Val Gly His Val Thr Thr Glu Val
Val Val Ala Tyr Thr 565 570
575 Thr Trp Cys Pro Val Glu Asn Gly Pro His Pro Thr Ala Pro Pro Lys
580 585 590 Pro Ala
Ala Pro Glu Ile Thr Ala Thr Phe Thr Leu Pro Asn Thr Tyr 595
600 605 Thr Cys Ser Gln Gly Lys Asn
Thr Cys Ser Asn Pro Lys Thr Ala Pro 610 615
620 Asn Val Ile Val Val Thr Pro Ile Val Thr Gln Thr
Ala Pro Val Val 625 630 635
640 Ile Pro Gly Ile Ala Ala Pro Thr Pro Thr Pro Ser Val Ala Ala Ser
645 650 655 Ser Pro Ala
Ser Pro Ser Val Val Pro Ser Pro Thr Ala Pro Val Ala 660
665 670 Thr Ser Pro Ala Gln Ser Ala Tyr
Tyr Pro Pro Pro Pro Pro Pro Glu 675 680
685 His Ala Val Ser Thr Pro Val Ala Asn Pro Pro Ala Val
Thr Pro Ala 690 695 700
Pro Ala Pro Phe Pro Ser Gly Gly Leu Thr Thr Val Ile Ala Pro Gly 705
710 715 720 Ser Thr Gly Val
Pro Ser Gln Pro Ala Gln Ser Gly Leu Pro Pro Val 725
730 735 Pro Ala Gly Ala Ala Gly Phe Arg Ala
Pro Ala Ala Val Ala Leu Leu 740 745
750 Ala Gly Ala Val Ala Ala Ala Leu Leu 755
760 136439PRTNeurospora crassa 136Met Val Ala Leu Thr Asn Leu
Leu Leu Thr Thr Leu Leu Ala Ser Ala 1 5
10 15 Gly Leu Gly Ala Ala Leu Pro Pro Arg Ile Gly
Ser Thr Val Ile Glu 20 25
30 Ala Arg Glu Pro Glu Leu Pro Val Ser Gly Arg Lys Ile Thr Leu
Pro 35 40 45 Gln
Gln Lys Asn Pro Arg Phe His Lys Phe Asn Gly Ala Leu Ser Val 50
55 60 Tyr Lys Thr Tyr Leu Lys
Tyr Gly Ala Pro Val Pro Asp His Leu Val 65 70
75 80 Gln Ala Val Ala Asn His Leu Gly Ile Ser Val
Glu Glu Val His Asn 85 90
95 Tyr Ala Asn Thr Thr Ala Asn Ala Arg Arg Asp Gln Gly Ser Ala Thr
100 105 110 Ala Ala
Pro Ile Asp Gln Ser Asp Ser Ala Tyr Ile Thr Pro Val Ser 115
120 125 Ile Gly Thr Pro Ala Gln Thr
Leu Asn Leu Asp Phe Asp Thr Gly Ser 130 135
140 Ser Asp Leu Trp Val Phe Ser Asn Ser Leu Pro Ser
Ser Gln Arg Ala 145 150 155
160 Gly His Thr Ile Tyr Asn Pro Ser Lys Ser Ser Thr Ala Lys Arg Val
165 170 175 Asn Gly Ala
Ser Trp Asp Ile Ser Tyr Gly Asp Gly Ser Ser Ser Lys 180
185 190 Gly Gln Val Tyr Leu Asp Lys Val
Thr Ile Gly Gly Leu Val Val Ser 195 200
205 Asn Gln Ala Val Glu Thr Ala Gln Gln Val Ser Gln Ser
Phe Thr Ala 210 215 220
Glu Thr Ser Ile Asp Gly Leu Val Gly Leu Ala Phe Gly Ser Leu Asn 225
230 235 240 Thr Val Arg Pro
Arg Gln Gln Lys Thr Trp Phe Glu Asn Ala Ile Gly 245
250 255 Gln Leu Asp Gln Pro Leu Phe Ala Ala
Asp Leu Lys Tyr Glu Ala Ser 260 265
270 Gly Thr Tyr Asp Phe Gly Phe Ile Asp Pro Ala Lys His Thr
Gly Asp 275 280 285
Ile Thr Tyr Val Pro Val Asn Thr Asn Pro Gly Tyr Trp Thr Trp Thr 290
295 300 Ser Thr Gly Tyr Gln
Val Gly Ser Ser Pro Phe Val Ser Gln Ser Ile 305 310
315 320 Thr Asn Ile Ala Asp Thr Gly Thr Thr Leu
Met Tyr Val Pro Asp Ser 325 330
335 Ile Leu Arg Ala Tyr Tyr Gly Gln Ile Arg Gly Ala Thr Asn Ser
Gln 340 345 350 Ser
Tyr Gly Gly Tyr Val Phe Pro Cys Ser Thr Glu Ala Pro Asp Phe 355
360 365 Thr Phe Gly Val Thr Asp
Glu Ala Thr Ile Thr Ile Pro Gly Arg Phe 370 375
380 Ile Asn Tyr Gly Pro Val Thr Asp Asp Gly Glu
Thr Cys Phe Gly Gly 385 390 395
400 Leu Gln Thr Ser Ser Asp Val Gly Ile Asn Ile Phe Gly Asp Val Ala
405 410 415 Leu Lys
Ala Ala Tyr Val Val Phe Lys Gly Gly Asp Ser Pro Ser Leu 420
425 430 Gly Trp Ala Ser Lys Gln Leu
435 137417PRTMyceliophthora thermophila 137Met
His Leu Thr Pro Ala Leu Val Ala Ala Thr Cys Ala Val Glu Val 1
5 10 15 Cys Ala Gly Val Leu Pro
Arg Ser Ser Ser Thr Pro Thr Thr Phe Gly 20
25 30 Ser Gly Thr Leu Ser Leu Lys Gln Val Arg
Asn Pro Asn Phe Val Arg 35 40
45 Asn Gly Pro Val Gln Leu Ala Arg Ile Tyr His Lys Tyr Gly
Val Pro 50 55 60
Leu Pro His Asp Leu Arg Glu Ala Val Ala Arg Phe Arg Ala Glu Ile 65
70 75 80 Arg Lys Arg Ser Asn
Gly Ser Thr Glu Thr Asn Pro Glu Thr Asn Asp 85
90 95 Val Glu Tyr Leu Thr Pro Val Ser Ile Gly
Thr Pro Pro Gln Val Leu 100 105
110 Asn Leu Asp Phe Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Ser
Ser 115 120 125 Glu
Thr Arg Ser Ser Asp Val Gln Gly Gln Thr Ile Tyr Asp Pro Asn 130
135 140 Glu Ser Ser Thr Ala Gln
Lys Leu Gln Gly Tyr Ser Trp Gln Ile Ser 145 150
155 160 Tyr Gly Asp Gly Ser Ser Ser Ser Gly Asp Val
Tyr Thr Asp Ala Val 165 170
175 Thr Val Gly Gly Leu Thr Val Pro Ser Gln Ala Val Glu Val Ala Arg
180 185 190 Arg Val
Ser Asp Glu Phe Thr Ser Asp Pro Asn Asn Asp Gly Leu Leu 195
200 205 Gly Leu Gly Phe Ser Ser Ile
Asn Thr Val Gln Pro Val Pro Gln Lys 210 215
220 Thr Phe Phe Asp Asn Ala Lys Ala Asp Leu Asp Ala
Pro Ile Phe Thr 225 230 235
240 Ala Asp Leu Lys Ala Ser Ala Pro Gly Phe Phe Asn Phe Gly Tyr Ile
245 250 255 Asp His Gly
Ala Tyr Thr Gly Glu Ile Thr Tyr Met Pro Val Asp Ser 260
265 270 Ser Asp Gly Phe Trp Ala Trp Thr
Ser Pro Gly Tyr Ala Val Gly Ser 275 280
285 Gly Ser Phe Lys Arg Thr Thr Ile Gln Gly Ile Ala Asp
Thr Gly Thr 290 295 300
Ser Leu Phe Leu Leu Pro Ser Ser Val Val Ser Ala Tyr Tyr Gly Gln 305
310 315 320 Ile Ser Gly Ala
Lys Tyr Asp Ser Ile Gln Gly Gly Tyr Thr Leu Pro 325
330 335 Cys Ser Gly Ser Val Pro Asp Phe Ala
Phe Gly Ile Gly Asp Ser Asn 340 345
350 Thr Thr Ile Ser Val Pro Gly Asp Tyr Val Arg Tyr Ala Ala
Thr Asp 355 360 365
Ser Ser Gly Ile Ile Cys Phe Gly Gly Ile Gln Ala Asn Thr Gly Ile 370
375 380 Gly Phe Ser Ile Phe
Gly Asp Val Ala Leu Lys Ala Ala Phe Val Val 385 390
395 400 Phe Asp Gly Ala Lys Gln Gln Leu Gly Trp
Ala Ser Lys Pro Leu Pro 405 410
415 Ser 138434PRTNeurospora crassa 138Met Leu Leu Phe Pro Thr
Ile Leu Thr Ala Ala Leu Ala Ala Thr Gly 1 5
10 15 Met Ala Ala Ala Ile Pro Ser Arg Asp Asp Thr
Thr Ala Asn Lys Gly 20 25
30 Thr Ala Ser Leu Leu Gln Val Arg Asn Pro Ser Phe Glu Phe Arg
His 35 40 45 Gly
Pro Leu Ala Leu Ala Lys Ala Tyr Gln Lys Phe Gly Ala Pro Met 50
55 60 Pro Glu Asp Leu Arg Ala
Ala Ile Ala Arg Phe Arg Gln Asn Gln Lys 65 70
75 80 Arg Thr Thr Gly Thr Ile Ala Thr Asp Pro Glu
Lys His Asp Val Glu 85 90
95 Tyr Leu Thr Pro Ile Ser Val Gly Thr Pro Ser Gln Asp Leu Val Val
100 105 110 Asp Phe
Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Thr Glu Met 115
120 125 Ser Thr Ser Asp Ile Lys Gly
Gln Thr Val Tyr Asp Pro Asn Asn Ser 130 135
140 Ser Thr Ser Glu Lys Val Gln Gly Ser Thr Trp Lys
Ile Thr Tyr Gly 145 150 155
160 Asp Gly Ser Ser Ser Ser Gly Asp Val Tyr Leu Asp Thr Val Thr Ile
165 170 175 Gly Asn Leu
Thr Val Pro Ser Gln Ala Val Glu Ala Ala Lys Lys Val 180
185 190 Ser Ser Glu Phe Thr Asp Asp Ser
His Asn Asp Gly Leu Leu Gly Leu 195 200
205 Gly Phe Ser Ala Ile Asn Ala Val Glu Pro Thr Pro Gln
Asn Thr Phe 210 215 220
Phe Asp Asn Ile Lys Gly Ser Leu Asp Ala Pro Leu Phe Thr Val Asp 225
230 235 240 Leu Lys His Gly
Thr Pro Gly Ser Phe Asn Phe Gly Tyr Ile Asp Pro 245
250 255 Ala Ala Tyr Ile Gly Asn Ile Ser Trp
Thr Pro Val Asp Ser Ser Gln 260 265
270 Gly Tyr Trp Gly Phe Thr Ser Pro Gly Tyr Ala Val Gly Thr
Gly Ala 275 280 285
Phe Arg Asn His Ser Ile Ser Gly Ile Ala Asp Thr Gly Thr Thr Leu 290
295 300 Leu Leu Leu Pro Lys
Ser Val Val Ser Ala Tyr Tyr Lys Glu Ile Gln 305 310
315 320 Gly Ala Gln Tyr Asp Ser Asp Gln Gly Gly
Tyr Ile Phe Pro Cys Ser 325 330
335 Pro Thr Pro Pro Asp Phe Val Phe Gly Val Asn Lys Gly Ile Val
Thr 340 345 350 Val
Pro Gly Asp Met Val Ser Tyr Ala Pro Ala Asp Ser Ala Asn Gln 355
360 365 Asn Cys Phe Gly Gly Ile
Gln Thr Asp Thr Gly Ile Gly Phe Ser Ile 370 375
380 Phe Gly Asp Val Ala Leu Lys Thr Ser Phe Val
His Leu His Gly Ser 385 390 395
400 Ile Val Pro Gly Tyr Tyr Ala Asp Cys Ala Met Arg Phe Asn Arg Met
405 410 415 Leu Arg
Ser Tyr Ser Asn Asp Gln Leu Val Asp Phe Ser Ser Ser Gly 420
425 430 Pro Leu
139466PRTMyceliophthora thermophila 139Met Asp Ala Leu Phe Glu Thr His
Ala Lys Leu Arg Lys Arg Met Ala 1 5 10
15 Leu Tyr Arg Val Arg Ala Val Pro Asn Gln Asn Tyr Gln
Arg Asp Gly 20 25 30
Thr Lys Ser Tyr Val Ser Val Leu Asn Arg Phe Gly Phe Gln Pro Thr
35 40 45 Lys Pro Gly Pro
Tyr Phe Gln Ile Phe Glu Glu Ser Glu Glu Ala Pro 50
55 60 Ser Met Ser Ala Ala Pro Gly Val
Lys Pro Gly His Val Trp Gln Gly 65 70
75 80 Leu Phe Lys Lys Leu Lys Asp Gln Glu Glu Pro Gly
Glu Val Thr Ala 85 90
95 Glu Asp Gln Gln Asn Asp Ser Glu Tyr Leu Cys Glu Val Met Ile Gly
100 105 110 Thr Ala Trp
Thr Ala Glu Arg Gln Ile Val Lys Met Asp Phe Asp Thr 115
120 125 Gly Leu Ala Asp Phe Trp Val Ser
Gln Lys Ser Phe Asp Pro Lys Lys 130 135
140 Ser Val Thr Trp Gln Leu Ala Lys Asp Lys Ser Trp Lys
Val Gln Tyr 145 150 155
160 Gly Asp Gly Ser Ser Ala Ser Gly Ile Val Gly His Asp Ile Leu Ile
165 170 175 Ile Gly Gly Ile
Gln Ile Lys Arg Gln Ala Ile Glu Ile Ala Thr Glu 180
185 190 Met Ser Ala Gln Phe Ser Glu Gly Thr
Met Asp Gly Ile Leu Gly Leu 195 200
205 Ala Phe Ser Lys Leu Asn Thr Val Gln Thr Asp Gly Lys Pro
Asp Pro 210 215 220
Gln Arg Thr Val Val Asp Asn Met Met Ala Gln Asp Asp Ile Pro Pro 225
230 235 240 Glu Ala Glu Leu Phe
Ser Thr Ala Leu Tyr Ser Asn Arg Glu Asp Asp 245
250 255 Gln Arg Ser Phe Tyr Thr Phe Gly Trp Ile
Asp Glu Asp Leu Val Lys 260 265
270 Ala Ser Gly Glu Glu Ile Val Trp Thr Asp Val Asp Asn Ser Glu
Gly 275 280 285 Phe
Trp Met Phe Ser Ser Glu His Val Thr Ile Asp Gly Gln Gln Val 290
295 300 Arg Ile Glu Gly Asn Lys
Ala Ile Ala Asp Thr Gly Thr Ser Leu Val 305 310
315 320 Leu Val Ser Asp Gln Val Cys Asp Ala Leu Tyr
Ala His Ile Pro Ser 325 330
335 Ala Glu Tyr Ser Glu Glu Tyr Gln Gly Trp Thr Phe Pro Gln Glu Thr
340 345 350 Glu Val
Asp Lys Leu Pro Glu Phe Ser Ile Ala Ile Gly Asp Lys Glu 355
360 365 Phe Val Leu Gln Lys Glu Asp
Leu Ile Phe Ala Pro Ala Asp Glu Arg 370 375
380 Val Phe Tyr Gly Ser Val Gln Ser Arg Gly Glu Asn
Pro Phe Asp Ile 385 390 395
400 Leu Gly Ile Ala Phe Leu Lys Ser Ile Tyr Ala Ile Trp Asp Gln Gly
405 410 415 His Lys Arg
Phe Gly Ala Val Pro Lys Met Glu Ala Phe Val Pro Pro 420
425 430 Thr Lys Tyr Asp Arg Pro Arg Leu
Thr Asp Gln Asp Arg Lys Asp Leu 435 440
445 Gly Val Thr Ile Gly Tyr Gly Asp Ile Ser Ser Thr Phe
Phe Glu Lys 450 455 460
Arg Ala 465 140481PRTNeurospora crassa 140Met Gly Ser Met Tyr Gln
Val Gln Ser Lys Leu Arg Gln Asp Leu Gly 1 5
10 15 Leu His Lys Val Gln Ala Val Arg Lys Pro Gly
Arg Glu Leu Asn Gly 20 25
30 Thr Lys Ala Tyr Val Ser Ala Met Ala Arg Tyr Gly Phe Asn Pro
Thr 35 40 45 Glu
Glu Ser Arg Phe Phe His Leu Lys Lys Thr Asp Leu Thr Lys Glu 50
55 60 Phe Gln Arg Arg Gly Tyr
Ile Arg His Trp Glu Gln Leu Val Arg Thr 65 70
75 80 Pro Gln Glu Arg Pro Asp Asp Pro His Thr Asp
Asn Glu Pro Val Pro 85 90
95 Ala Glu Asp Gln Gln Tyr Asp Thr Gln Tyr Leu Cys Glu Ile Gly Ile
100 105 110 Gly Thr
Pro Gln Gln Lys Val Lys Leu Asp Phe Asp Thr Gly Ser Ala 115
120 125 Asp Leu Trp Val Arg Cys Thr
Asp Ser Ser Leu Leu His His Ala Asp 130 135
140 Lys Lys Phe Asp Pro Lys Lys Ser Asp Thr Phe Gln
Glu Ser Lys Thr 145 150 155
160 Asp Gln Thr Trp Lys Ile Gln Tyr Gly Asp Gly Ser Thr Ala Ser Gly
165 170 175 Thr Val Gly
Thr Asp Val Ile Thr Val Gly Gly Leu Gln Ile Lys Asn 180
185 190 Gln Ala Ile Glu Leu Ala Lys Lys
Val Ser Ser Ala Phe Ser Ser Gly 195 200
205 Glu Ala Asp Gly Leu Leu Gly Leu Ala Phe Ser Thr Ile
Asn Thr Ile 210 215 220
Glu Ser Asp Gly Lys Pro Asp Pro Gln Pro Thr Pro Val Glu Asn Met 225
230 235 240 Ile Ser Gln Glu
Asp Ile Pro Lys Glu Ala Glu Leu Phe Thr Ser Ala 245
250 255 Phe Tyr Ser Ala Arg Asp Asp Lys Ser
Glu Glu Lys Ser Phe Tyr Thr 260 265
270 Phe Gly Trp Val Asp Glu Asp Leu Val Lys Ala Ser Gly Lys
Asp Ile 275 280 285
Thr Trp Thr Pro Ile Asp Asn Ser Glu Gly Phe Trp Lys Phe Pro Ser 290
295 300 Glu Ser Ala Thr Val
Asp Gly Asp Asn Val Ser Val Ser Gly Asn Thr 305 310
315 320 Ala Ile Ala Asp Thr Gly Thr Thr Leu Ala
Leu Val Ser Asp Thr Val 325 330
335 Cys Lys Ala Leu Tyr Ala Lys Ile Pro Gly Ser Lys Tyr Ser Tyr
Arg 340 345 350 Tyr
Gln Gly Tyr Leu Ile Pro Ser Thr Ile Thr Ala Asp Gln Leu Pro 355
360 365 Gln Leu Ser Val Ala Val
Gly Gly Glu Gln Phe Val Ile Gln Asn Glu 370 375
380 Asp Leu Leu Leu Ala Pro Ala Asp Asp Asp His
Trp Tyr Gly Gly Val 385 390 395
400 Gln Ser Arg Gly Thr Met Pro Phe Asp Ile Leu Gly Asp Thr Phe Leu
405 410 415 Lys Ser
Ile Tyr Ala Ile Trp Asp Gln Gly Asn Asn Arg Phe Gly Ala 420
425 430 Val Pro Lys Ile Glu Val Asn
Gln His Thr Val Phe Pro Asp Thr Glu 435 440
445 Pro Ser Pro Glu Ala Ser Ser Pro Glu Pro Ala Asp
Lys Val Gly Asp 450 455 460
Val Ser Pro Val Glu Gln Val Lys Gly Ala Val Lys Ser Leu Lys Val 465
470 475 480 Leu
141397PRTMyceliophthora thermophila 141Met Lys Asp Ala Phe Leu Leu Thr
Ala Ala Val Leu Leu Gly Ser Ala 1 5 10
15 Gln Gly Ala Val His Lys Met Lys Leu Gln Lys Ile Pro
Leu Ser Glu 20 25 30
Gln Leu Glu Ala Val Pro Ile Asn Thr Gln Leu Glu His Leu Gly Gln
35 40 45 Lys Tyr Met Gly
Leu Arg Pro Arg Glu Ser Gln Ala Asp Ala Ile Phe 50
55 60 Lys Gly Met Val Ala Asp Val Lys
Gly Asn His Pro Ile Pro Ile Ser 65 70
75 80 Asn Phe Met Asn Ala Gln Tyr Phe Ser Glu Ile Thr
Ile Gly Thr Pro 85 90
95 Pro Gln Ser Phe Lys Val Val Leu Asp Thr Gly Ser Ser Asn Leu Trp
100 105 110 Val Pro Ser
Val Glu Cys Gly Ser Ile Ala Cys Tyr Leu His Ser Lys 115
120 125 Tyr Asp Ser Ser Ala Ser Ser Thr
Tyr Lys Lys Asn Gly Thr Ser Phe 130 135
140 Glu Ile Arg Tyr Gly Ser Gly Ser Leu Ser Gly Phe Val
Ser Gln Asp 145 150 155
160 Thr Val Ser Ile Gly Asp Ile Thr Ile Gln Gly Gln Asp Phe Ala Glu
165 170 175 Ala Thr Ser Glu
Pro Gly Leu Ala Phe Ala Phe Gly Arg Phe Asp Gly 180
185 190 Ile Leu Gly Leu Gly Tyr Asp Arg Ile
Ser Val Asn Gly Ile Val Pro 195 200
205 Pro Phe Tyr Lys Met Val Glu Gln Lys Leu Ile Asp Glu Pro
Val Phe 210 215 220
Ala Phe Tyr Leu Ala Asp Thr Asn Gly Gln Ser Glu Val Val Phe Gly 225
230 235 240 Gly Val Asp His Asp
Lys Tyr Lys Gly Lys Ile Thr Thr Ile Pro Leu 245
250 255 Arg Arg Lys Ala Tyr Trp Glu Val Asp Phe
Asp Ala Ile Ser Tyr Gly 260 265
270 Asp Asp Thr Ala Glu Leu Glu Asn Thr Gly Ile Ile Leu Asp Thr
Gly 275 280 285 Thr
Ser Leu Ile Ala Leu Pro Ser Gln Leu Ala Glu Met Leu Asn Ala 290
295 300 Gln Ile Gly Ala Lys Lys
Ser Tyr Thr Gly Gln Tyr Thr Ile Asp Cys 305 310
315 320 Asn Lys Arg Asp Ser Leu Lys Asp Val Thr Phe
Asn Leu Ala Gly Tyr 325 330
335 Asn Phe Thr Leu Gly Pro Tyr Asp Tyr Val Leu Glu Val Gln Gly Ser
340 345 350 Cys Ile
Ser Thr Phe Met Gly Met Asp Phe Pro Ala Pro Thr Gly Pro 355
360 365 Leu Ala Ile Leu Gly Asp Ala
Phe Leu Arg Arg Tyr Tyr Ser Ile Tyr 370 375
380 Asp Leu Gly Ala Asp Thr Val Gly Leu Ala Glu Ala
Lys 385 390 395
142396PRTNeurospora crassa 142Met Lys Gly Ala Leu Leu Thr Ala Ala Met Leu
Leu Gly Ser Ala Gln 1 5 10
15 Ala Gly Val His Thr Met Lys Leu Lys Lys Val Pro Leu Ala Glu Gln
20 25 30 Leu Glu
Ser Val Pro Ile Asp Val Gln Val Gln His Leu Gly Gln Lys 35
40 45 Tyr Thr Gly Leu Arg Thr Glu
Ser His Thr Gln Ala Met Phe Lys Ala 50 55
60 Thr Asp Ala Gln Val Ser Gly Asn His Pro Val Pro
Ile Thr Asn Phe 65 70 75
80 Met Asn Ala Gln Tyr Phe Ser Glu Ile Thr Ile Gly Thr Pro Pro Gln
85 90 95 Thr Phe Lys
Val Val Leu Asp Thr Gly Ser Ser Asn Leu Trp Val Pro 100
105 110 Ser Ser Gln Cys Gly Ser Ile Ala
Cys Tyr Leu His Asn Lys Tyr Glu 115 120
125 Ser Ser Glu Ser Ser Thr Tyr Lys Lys Asn Gly Thr Ser
Phe Lys Ile 130 135 140
Glu Tyr Gly Ser Gly Ser Leu Ser Gly Phe Val Ser Gln Asp Arg Met 145
150 155 160 Thr Ile Gly Asp
Ile Thr Ile Asn Asp Gln Leu Phe Ala Glu Ala Thr 165
170 175 Ser Glu Pro Gly Leu Ala Phe Ala Phe
Gly Arg Phe Asp Gly Ile Leu 180 185
190 Gly Leu Gly Tyr Asp Arg Ile Ala Val Asn Gly Ile Thr Pro
Pro Phe 195 200 205
Tyr Lys Met Val Glu Gln Lys Leu Val Asp Glu Pro Val Phe Ser Phe 210
215 220 Tyr Leu Ala Asp Gln
Asp Gly Glu Ser Glu Val Val Phe Gly Gly Val 225 230
235 240 Asn Lys Asp Arg Tyr Thr Gly Lys Ile Thr
Thr Ile Pro Leu Arg Arg 245 250
255 Lys Ala Tyr Trp Glu Val Asp Phe Asp Ala Ile Gly Tyr Gly Lys
Asp 260 265 270 Phe
Ala Glu Leu Glu Gly His Gly Val Ile Leu Asp Thr Gly Thr Ser 275
280 285 Leu Ile Ala Leu Pro Ser
Gln Leu Ala Glu Met Leu Asn Ala Gln Ile 290 295
300 Gly Ala Lys Lys Ser Trp Asn Gly Gln Phe Thr
Ile Asp Cys Gly Lys 305 310 315
320 Lys Ser Ser Leu Glu Asp Val Thr Phe Thr Leu Ala Gly Tyr Asn Phe
325 330 335 Thr Leu
Gly Pro Glu Asp Tyr Ile Leu Glu Ala Ser Gly Ser Cys Leu 340
345 350 Ser Thr Phe Met Gly Met Asp
Met Pro Ala Pro Val Gly Pro Leu Ala 355 360
365 Ile Leu Gly Asp Ala Phe Leu Arg Lys Tyr Tyr Ser
Ile Tyr Asp Leu 370 375 380
Gly Ala Asp Thr Val Gly Ile Ala Thr Ala Lys Arg 385
390 395 143454PRTMyceliophthora thermophila 143Met
Lys Phe Ala Ala Leu Ala Leu Ala Ala Ser Leu Val Ala Ala Ala 1
5 10 15 Pro Arg Val Val Lys Val
Asp Pro Ser Asp Ile Lys Pro Arg Arg Leu 20
25 30 Gly Gly Thr Lys Phe Lys Leu Gly Gln Ile
His Asn Asp Leu Phe Arg 35 40
45 Gln His Gly Arg Gly Pro Arg Ala Leu Ala Lys Ala Tyr Glu
Lys Tyr 50 55 60
Asn Ile Glu Leu Pro Pro Asn Leu Leu Glu Val Val Gln Arg Ile Leu 65
70 75 80 Lys Asp Leu Gly Ile
Glu Pro His Ser Lys Lys Ile Pro Gly Ser Lys 85
90 95 Ser Ser Tyr Gly Asn Gly Ala Pro Tyr Thr
Asn Glu Thr Asp Asp Ser 100 105
110 Gly Glu Val Ser Ala Ile Pro Gln Leu Phe Asp Val Glu Tyr Leu
Ala 115 120 125 Pro
Val Gln Ile Gly Thr Pro Pro Gln Thr Leu Met Leu Asn Phe Asp 130
135 140 Thr Gly Ser Ser Asp Leu
Trp Val Phe Ser Ser Glu Thr Pro Ser Arg 145 150
155 160 Gln Gln Asn Gly Gln Lys Ile Tyr Lys Ile Glu
Glu Ser Ser Thr Ala 165 170
175 Arg Arg Leu Ser Asn His Thr Trp Ser Ile Gln Tyr Gly Asp Gly Ser
180 185 190 Arg Ser
Ala Gly Asn Val Tyr Leu Asp Thr Val Ser Val Gly Gly Val 195
200 205 Asn Val Phe Asn Gln Ala Val
Glu Ser Ala Thr Phe Val Ser Ser Ser 210 215
220 Phe Val Thr Asp Ala Ala Ser Ser Gly Leu Leu Gly
Leu Gly Phe Asp 225 230 235
240 Ser Ile Asn Thr Val Lys Pro Thr Lys Gln Lys Thr Phe Ile Ser Asn
245 250 255 Ala Leu Glu
Ser Leu Glu Met Gly Leu Phe Thr Ala Asn Leu Lys Lys 260
265 270 Ala Glu Pro Gly Asn Tyr Asn Phe
Gly Phe Ile Asp Glu Thr Glu Phe 275 280
285 Val Gly Pro Leu Ser Phe Ile Asp Val Asp Ser Thr Asp
Gly Phe Trp 290 295 300
Gln Phe Asp Ala Thr Gly Tyr Ser Ile Gln Leu Pro Glu Pro Ser Gly 305
310 315 320 Asn Ile Thr Gly
Thr Pro Phe Arg Ala Val Ala His Thr Ala Ile Ala 325
330 335 Asp Thr Gly Thr Thr Leu Leu Leu Leu
Pro Pro Gly Ile Ala Gln Ala 340 345
350 Tyr Tyr Trp Gln Val Gln Gly Ala Arg Gln Ala Pro Glu Val
Gly Gly 355 360 365
Trp Val Met Pro Cys Asn Ala Ser Met Pro Asp Leu Thr Leu His Ile 370
375 380 Gly Thr Tyr Lys Ala
Val Ile Pro Gly Glu Leu Ile Pro Tyr Ala Pro 385 390
395 400 Val Asp Thr Asp Asp Met Asp Thr Ala Thr
Val Cys Tyr Gly Gly Ile 405 410
415 Gln Ser Ala Ser Gly Met Pro Phe Ala Ile Tyr Gly Asp Ile Phe
Phe 420 425 430 Lys
Ala Gln Phe Thr Val Phe Asp Val Glu Asn Leu Lys Leu Gly Phe 435
440 445 Ala Pro Lys Pro Glu Leu
450 144489PRTMyceliophthora thermophila 144Met Ala
Ile Pro Val Leu Phe Ser Ala Leu Val Leu Leu Val Ala Leu 1 5
10 15 Leu Cys Cys His Ala Thr Ala
Ala Leu Gln Gln Leu Ala His Asp Val 20 25
30 Gly Cys Val His Leu Pro Val Val His Ser Thr Lys
Val Asp Arg Phe 35 40 45
Ser Asp Lys Arg Gly Ile Gln Leu Gln Leu Ala Asn Arg Ser Asp Val
50 55 60 Ala Tyr Tyr
Ala Gln Leu Ser Ile Gly Thr Pro Pro Gln Pro Val Phe 65
70 75 80 Val Gln Leu Asp Thr Gly Ser
Phe Glu Leu Trp Val Asn Pro Asp Cys 85
90 95 Thr Thr Val Ser Gly Ser Asp Ala Val Phe Cys
Glu Arg Ala Gly Arg 100 105
110 Tyr Asp Val Thr Lys Ser Ser Thr Ala Thr Ser Leu Gly Thr Asn
Arg 115 120 125 Thr
Leu Arg Tyr Gly Ile Gly Ala Ala Asn Ile Ser Tyr Phe Thr Asp 130
135 140 Thr Ile Ser Leu Ala Gly
Ser Pro Met Met Leu Gln Asp Val Gln Phe 145 150
155 160 Gly Val Ala Thr Ala Ser Glu Asp Ala Phe Ser
Gly Ile Leu Gly Ile 165 170
175 Gly Tyr Gly Lys Gly Ile Gly Thr Gly Tyr Pro Asn Phe Val Asp Gln
180 185 190 Leu Trp
Glu Gln Asn Val Thr Arg Val Lys Ala Tyr Thr Leu Ala Leu 195
200 205 Gly Ser Lys Asp Ser Gln Glu
Gly Val Ile Val Phe Gly Gly Val Asp 210 215
220 Thr Ser Lys Phe Ala Gly Lys Leu Ala Arg Leu Pro
Val Ile Pro Pro 225 230 235
240 Ala Gln Ser Pro Asp Gly Val Pro Arg Phe Trp Val Glu Met Lys Ser
245 250 255 Leu Ser Ile
Thr Arg Pro Ser Gly Leu Asn Thr Val Tyr Asp Gly Gly 260
265 270 Ala Met Pro Val Phe Leu Asp Ser
Gly Ser Thr Met Thr Leu Leu Pro 275 280
285 Ala Asn Leu Thr Met Ala Val Ala Arg Asp Phe Gly Ala
Gln Ala Pro 290 295 300
Asp Ala Asn Gly Phe Tyr Lys Ile Asp Cys Ala Leu Thr Ala Leu Asn 305
310 315 320 Gly Thr Leu Asp
Phe Ala Phe Asp Gly Val Thr Val Arg Val Pro Tyr 325
330 335 Lys Glu Leu Thr Arg Glu Val Ala Ser
Asn Pro Pro Ser Cys Phe Leu 340 345
350 Gly Ile Val Ala Ser Asp Arg Phe Thr Leu Leu Gly Asp Thr
Phe Leu 355 360 365
Arg Ser Ala Tyr Thr Val Phe Asp Leu Glu Thr Asp Ser Ile Trp Met 370
375 380 Ala Pro Ala Val Asn
Cys Gly Ser Ser Pro Ala Ala Leu Ser Asn Val 385 390
395 400 Gln Asp Leu Ser Ala Val Thr Gly Glu Cys
Gly Val Arg Glu Ile Ala 405 410
415 Glu Ser Thr Ser Ser Thr Gln Val Pro Ser Thr Gly Val Asp Asp
Thr 420 425 430 Glu
Ala Gly Ala Val Pro Thr Ser Thr Thr Thr Val Val Ser Gln Pro 435
440 445 Ser Gly Thr Thr Thr Gln
Met Gly Ala Arg Pro Thr Leu Asp Asn Ala 450 455
460 Ser Asn Pro Leu Gly Ala His Arg Leu Thr Trp
Val Leu Val Ile Thr 465 470 475
480 Ala Ala Leu His Leu Phe Thr Gly Ile 485
145518PRTNeurospora crassa 145Met Ala Ala Phe Pro Phe Leu Ser Ala
Ser Phe Val Leu Leu Gln Leu 1 5 10
15 Ala Leu Thr Cys Leu Ala Gln His Leu Asn Leu Thr Thr Gly
Pro Leu 20 25 30
His Leu Thr Gly His Thr Pro Gly Asp Gly Cys Val His Leu Pro Ile
35 40 45 Ile His Ser Thr
Asn Thr Asp His Phe Ala Arg Arg Gly Ile Gln Leu 50
55 60 Ala Leu Asn Asn Arg Ser Asp Val
Ala Tyr Tyr Ala Gln Leu Glu Ile 65 70
75 80 Gly Thr Pro Pro Gln Thr Val Tyr Thr Gln Leu Asp
Thr Gly Ser Phe 85 90
95 Glu Leu Trp Val Asn Pro Asp Cys Thr Thr Val Ser Pro Ser Asp Ser
100 105 110 Ser Phe Cys
Asp His Ile Gly Phe Tyr Asn Ala Ser Leu Ser Ser Thr 115
120 125 Ser Lys Ser Leu Gly Thr Ser Lys
Thr Leu Arg Tyr Gly Ile Gly Ala 130 135
140 Ala Asn Ile Ser Tyr Val Thr Asp Thr Ile Ser Leu Ser
Gly Ser Ser 145 150 155
160 Thr Ser Leu Lys Asp Ile Gln Phe Gly Val Ala Thr Ser Ser Lys Asp
165 170 175 Ala Phe Ser Gly
Ile Leu Gly Ile Gly Tyr Gly Gln Gly Leu Ala Thr 180
185 190 Lys Tyr Pro Asn Phe Ile Asp Gln Leu
Tyr Ala Gln Lys Ile Thr Lys 195 200
205 Val Lys Ala Tyr Thr Leu Ala Leu Gly Ser Lys Thr Ala Gln
Gln Gly 210 215 220
Ser Ile Val Phe Gly Gly Val Asp Thr Ser Lys Phe Ala Gly Pro Leu 225
230 235 240 Gly Arg Leu Pro Ile
Ile Pro Ala Glu Asp Ser Pro Asp Gly Val Pro 245
250 255 Arg Phe Trp Val Gln Met Asn Gly Ile Ser
Leu Thr Pro Pro Ser Gly 260 265
270 Gln Ser Met Gly Val Tyr Glu Gly Ser Lys Ile Pro Ala Phe Leu
Asp 275 280 285 Ser
Gly Ser Thr Met Thr Ile Leu Pro Pro Ala Leu Ala Asn Lys Ile 290
295 300 Ala Glu Asp Phe Gly Ser
Pro Glu Met Asp Ala Asn Gly Phe Tyr Arg 305 310
315 320 Val Gly Cys Gly Tyr Val Glu Met Asn Gly Thr
Met Asp Phe Glu Phe 325 330
335 Val Gly Ala Gly Gln Lys Val Thr Val Arg Val Pro Tyr Lys Glu Met
340 345 350 Ile Arg
Glu Val Gly Gln Gly Glu Ser Lys Met Cys Phe Leu Gly Ile 355
360 365 Met Gly Ser Glu Ser Phe Thr
Leu Leu Gly Asp Thr Phe Leu Arg Ser 370 375
380 Ala Tyr Ala Thr Ser Cys Gly Asn Thr Pro Ala Ala
Leu Arg Asp Val 385 390 395
400 Thr Asp Leu Ser Arg Val Val Gly Asn Cys Gln Ile Gln Leu Gly Glu
405 410 415 Lys Glu Ala
Val Val Asp Val Val Ser Glu Thr Ser Ile Ala Pro Pro 420
425 430 Thr Gly Ser Thr Gly Asp Thr Asp
Gly Val Thr Gly Thr Gly Gly Asn 435 440
445 Gly Ser Gly Asn Gly Gly Thr Arg Thr Ala Trp Gly Phe
Val Thr Thr 450 455 460
Thr Leu Ala Val Pro Met Ala Thr Gly Leu Ala Gly Val Gly Gly Ser 465
470 475 480 Gly Ser Gly Ser
Met Ser Ala Thr Ala Leu Asp Ser Ser Gly Arg Ser 485
490 495 Met Ala Gly Asp Val Val Leu Ser Ala
Ala Val Ala Val Gly Ala Ala 500 505
510 Val Leu Gly Ser Leu Leu 515
146501PRTMyceliophthora thermophila 146Met Arg Gly Tyr Ala Ala Val Ala
Phe Gly Ala Ile Leu Ala Gly Ala 1 5 10
15 Val His Ala Ser Ala Gly Asn Gly Val Val Gln Trp Asp
Ile Arg Arg 20 25 30
Thr Gln Arg Gln Glu Glu Leu Gln Arg Leu Asn Arg Arg Leu Arg Lys
35 40 45 Arg Ala Asn Pro
Val Leu Glu Val Ile Thr Asn Glu Lys Ile Arg Gly 50
55 60 Gly Tyr Phe Ala Thr Cys Lys Ile
Gly Thr Pro Gly Gln Asp Leu Thr 65 70
75 80 Leu Gln Leu Asp Thr Gly Ser Ser Asp Ile Trp Val
Pro Asp Ser Ala 85 90
95 Ala Gln Val Cys Arg Glu Ile Gly Thr Glu Gly Cys Ala Leu Gly Thr
100 105 110 Phe Asn Pro
Asn Arg Ser Ser Ser Phe Glu Val Ile Gly Glu Gly Gln 115
120 125 Phe Asp Ile Glu Tyr Val Asp Gly
Ser Ser Ser Lys Gly Asp Tyr Phe 130 135
140 Thr Asp Val Phe Gln Ile Gly Asp Ile Ser Val Gln Asn
Met Thr Met 145 150 155
160 Gly Leu Gly Leu His Thr Asp Ile Ala Tyr Gly Leu Val Gly Val Gly
165 170 175 Tyr Ala Ile Asn
Glu Ala Ile Val Ala Thr Thr Gln Ser Arg Asp Ser 180
185 190 Val Tyr Pro Asn Leu Pro Val Gln Met
Val Asp Gln Gly Leu Ile Asn 195 200
205 Thr Val Ala Tyr Ser Leu Trp Leu Asn Asp Leu Asp Ala Ser
Ser Gly 210 215 220
Ser Ile Leu Phe Gly Gly Ile Asp Thr Glu Lys Tyr Gln Gly Glu Leu 225
230 235 240 Thr Arg Ile Asp Ile
Tyr Pro Thr Ser Gln Gly Asp Phe Ser Ser Phe 245
250 255 Val Val Ala Leu Thr Ser Leu Glu Ala Arg
Ser Pro Ser Gly Gln Asp 260 265
270 Thr Leu Thr Ser Gln Glu Phe Pro Ile Pro Val Val Leu Asp Ser
Gly 275 280 285 Thr
Thr Leu Ser Tyr Leu Pro Thr Asp Leu Ala Thr Gln Ala Trp Lys 290
295 300 Glu Val Gly Ala Phe Tyr
Leu Pro Glu Val Gly Ala Ala Val Leu Pro 305 310
315 320 Cys Asp Met Glu Asn Ser Lys Gly Ser Phe Ser
Phe Gly Phe Ala Gly 325 330
335 Pro Asp Gly Pro Arg Ile Thr Val Gly Met Asp Glu Leu Val Leu Asp
340 345 350 Met Thr
Asp Gly Gln Ala Pro Gln Phe Leu Ser Gly Pro Tyr Lys Gly 355
360 365 Arg Asp Val Cys Gln Phe Gly
Ile Gln Asn Phe Thr Ser Ala Pro Phe 370 375
380 Leu Leu Gly Asp Thr Phe Leu Arg Ser Ala Tyr Val
Val Tyr Asp Leu 385 390 395
400 Val Asn Asn Gln Ile Gly Ile Ala Ala Thr Asp Phe Asn Ser Thr Asp
405 410 415 Ser Asn Ile
Val Pro Phe Pro Ser Met Gly Ala Pro Ile Pro Ser Ala 420
425 430 Thr Val Ala Ala Asn Gln Arg Glu
Val Thr Arg Val Pro Thr Val Thr 435 440
445 Glu Pro Ala Tyr Ser Ala Ser Gln Gly Phe Met Glu Ser
Ala Ser Gly 450 455 460
Glu Glu Ser Leu Ala Pro Gly Met Pro Ala Ala Trp Gly Met Gly Gln 465
470 475 480 Leu Leu Val Val
Gly Val Thr Met Ala Leu Thr Ala Leu Gly Ser Gly 485
490 495 Leu Phe Phe Val Leu 500
147492PRTNeurospora crassa 147Met Lys Gly Tyr Thr Ser Ser Ala Leu Leu
Leu Gly Pro Ala Leu Leu 1 5 10
15 Ser Gln Leu Ala Leu Ala Gln Gln Ala Pro Asn Gly Val Val His
Trp 20 25 30 Gly
Ile Gln Lys Arg His Ala Pro Asn Ala Pro Asn Arg Leu Leu Arg 35
40 45 Arg Ala Gly Pro Thr His
Gln Ala Ile Leu Gln Asn Glu Gln Ala Arg 50 55
60 Gly Gly Tyr Phe Ala Thr Cys Ala Met Gly Thr
Pro Gly Gln Lys Val 65 70 75
80 Thr Leu Gln Leu Asp Thr Gly Ser Ser Asp Val Trp Val Pro Asp Ser
85 90 95 Thr Ala
Ser Ile Cys Asn Lys Gly Ala Cys Asp Leu Gly Ser Trp Gln 100
105 110 Gly Glu Phe Asp Ile Ser Tyr
Val Asp Gly Ser Ser Ser Lys Gly Asp 115 120
125 Tyr Phe Thr Asp Val Phe Asn Ile Gly Gly Thr Thr
Val Thr Asn Leu 130 135 140
Thr Met Gly Leu Gly Ala Gln Thr Asp Ile Ala Tyr Gly Leu Val Gly 145
150 155 160 Ile Gly Tyr
Ala Ile Asn Glu Ala Ile Val Gly Asn Ser His Ser Leu 165
170 175 Ser Ser Gln Tyr Pro Asn Leu Pro
Val Ala Met Val Asp Asp Gly Leu 180 185
190 Ile Asn Thr Ile Ala Tyr Ser Leu Trp Leu Asn Asp Leu
Asp Ala Gly 195 200 205
Glu Gly Ser Ile Leu Phe Gly Gly Ile Asp Thr Lys Lys Tyr Lys Gly 210
215 220 Asp Leu Thr Arg
Ile Arg Ile Tyr Pro Ser Ser Asn Gly Tyr Tyr Phe 225 230
235 240 Ser Phe Ile Val Ala Leu Thr Ser Leu
Gln Ala Ile Ser Pro Ser Gly 245 250
255 Asn Asp Thr Leu Thr Ser Gln Glu Phe Pro Ile Pro Val Val
Leu Asp 260 265 270
Ser Gly Thr Thr Leu Ser Tyr Leu Pro Gln Asp Ile Val Asp Gln Ile
275 280 285 Trp Gln Glu Val
Gly Ala Glu Tyr Ser Asp Arg Leu Glu Leu Ala Val 290
295 300 Ile Pro Cys Ser Lys Lys Ser Ser
Asn Gly Tyr Phe Ser Phe Gly Phe 305 310
315 320 Ala Gly Pro Asp Gly Pro Arg Ile Thr Val Arg Met
Asp Glu Leu Val 325 330
335 Leu Asp Leu Thr Ser Gly Asp Pro Pro Lys Tyr Thr Ser Gly Pro Asn
340 345 350 Lys Gly Gln
Asp Val Cys Glu Phe Gly Ile Gln Asn Ser Thr Ser Ala 355
360 365 Pro Tyr Leu Leu Gly Asp Thr Phe
Leu Arg Ser Ala Tyr Val Val Tyr 370 375
380 Asp Leu Val Asn Asn Glu Ile Gly Leu Ala Glu Thr Asp
Phe Asn Ser 385 390 395
400 Thr Glu Ser Asn Ile Val Ala Phe Ala Ser Met Ser Ala Thr Ile Pro
405 410 415 Ser Ala Thr Gln
Ala Pro Asn Gln Ala Ala Val Thr Asn Arg Pro Val 420
425 430 Ala Thr Met Pro Ser Phe Ala Ala Ser
Ser Gly Phe Ser Asp Thr Gly 435 440
445 Gly Ser Gly Asn Asp Gly Lys Asp Glu Asn Ala Ser Ala Gly
Met Pro 450 455 460
Ser Ala Phe Gly Val Ala Gln Met Ser Val Met Gly Ile Ala Met Val 465
470 475 480 Phe Ala Met Val Gly
Ser Gly Val Phe Val Leu Leu 485 490
148668PRTMyceliophthora thermophila 148Met Ser Phe Ala Leu Tyr Ala Ala
Ala Leu Leu Pro Val Ala Val Leu 1 5 10
15 Gly Ala Gly Leu Ser Val Pro Glu Asp Asn Arg Met Val
Gln Gln Asp 20 25 30
Gly Leu Leu Arg Tyr Pro Leu Met Pro Arg Leu Gly Asn Leu Leu Phe
35 40 45 Gly Lys His Ala
Asn Ile Thr Arg Arg Gln Ile Asp Thr Gly Ile Phe 50
55 60 Asp Pro Leu Ser Gly Thr Leu Tyr
Thr Ile Glu Leu Thr Leu Gly Thr 65 70
75 80 Pro Gly Gln Thr Val Pro Val Gln Phe Asp Thr Gly
Ser Asp Met Leu 85 90
95 Trp Val Asn Pro Val Cys Ser Lys Ala Ala Glu Pro Glu Phe Cys Ala
100 105 110 Ala Gln Pro
Arg Phe Thr Asp Ser Ser Thr Leu Val Asp Phe Gly Glu 115
120 125 Gln Gly Asn Ile Thr Tyr Gly Thr
Gly Tyr Ala Tyr Tyr Glu Tyr Val 130 135
140 Ala Asp Tyr Val Ala Ile Gly Ser Ala Arg Ile Thr Gln
Gln Val Phe 145 150 155
160 Gly Val Ala Leu Asp Ser Ala His Ala Asp Val Gly Ile Phe Gly Ala
165 170 175 Gly Pro Asn Leu
Asp Gly Trp Asp Ser Ala Tyr Pro Leu Val Val Asp 180
185 190 Ser Leu Ala Gln Gln Gly Tyr Thr Ser
Ser Arg Ala Phe Ser Met Asp 195 200
205 Leu Lys Gly Phe Glu Ser Ala Arg Gly Ser Val Ile Phe Gly
Gly Ile 210 215 220
Asp Thr Lys Lys Tyr Arg Gly Ser Leu Ile Lys Arg Leu Ile Ile Pro 225
230 235 240 Ala Ala Glu Ser Pro
Asp Gly Tyr Thr Arg Phe Trp Ile Tyr Leu Asp 245
250 255 Gly Ile Ser Val Asn Gln Pro Asp Gly Asp
Val Val Thr Val Phe Ser 260 265
270 Thr Pro Asp Gly Gly Lys Gly Gln Pro Val Leu Leu Asp Ser Gly
Tyr 275 280 285 Thr
Leu Ser Ala Leu Pro Arg Pro Ile Phe Gln Lys Leu Val Ala Ala 290
295 300 Phe Pro Ser Ala Gln Tyr
Val Ser Ser Ala Asp Val Tyr Val Val Asp 305 310
315 320 Cys Val Asp His Gly Glu Gly Gly Ser Leu Asp
Phe Ile Phe Gly Gly 325 330
335 Lys Thr Ile Asn Val Pro Tyr His Glu Phe Val Trp Ala Gln Pro Glu
340 345 350 Ser Asn
Thr Cys Val Leu Gly Ala Phe Glu Asp Asp Phe Pro Val Leu 355
360 365 Gly Asp Thr Phe Leu Arg Ser
Ala Tyr Val Val Tyr Asp Trp Asp Asn 370 375
380 Arg Asn Ile Tyr Leu Ala Gln Ser Asp Asp Cys Gly
Ser Asn Leu Val 385 390 395
400 Ala Ile Gly Ser Gly Pro Asp Ala Val Pro Ser Ile Val Gly Glu Cys
405 410 415 Gly Lys Pro
Lys Pro Thr Ser Thr Ser Thr Phe Ser Lys Thr Ser Ser 420
425 430 Lys Thr Ser Thr Ala Ser Lys Thr
Ser Ser Thr Ser Asp Ser Thr Ser 435 440
445 Ser Ser Ser Ser His Val Thr Thr Ser Ser Ser Ser Thr
Thr Ala Thr 450 455 460
Thr Leu Ser Thr His Lys Pro Pro Phe Pro Thr Ala Ser Gly Asn Phe 465
470 475 480 Thr Thr Thr Arg
Ser Pro Thr Thr Thr Thr Ala Ser Ser Thr Ile Ser 485
490 495 Lys Ser Thr Leu Thr Ile Thr Ser Ala
Thr Thr Tyr Thr Ile Thr Ser 500 505
510 Cys Pro Pro Thr Val Thr Arg Cys Pro Ala His Glu Val Thr
Thr Glu 515 520 525
Ile Ile Thr Lys Thr Thr Ala Val Cys Pro Glu Thr Thr Ala Thr Tyr 530
535 540 Thr Ile Pro Arg Thr
Ile Thr Cys Pro Gly Ser Gly Gly Gly Asp Asp 545 550
555 560 Cys Pro Pro Gly Ala Thr Arg Thr Thr Thr
Leu Thr Val Thr Leu Ser 565 570
575 Pro Val Gly Pro Thr Asp Arg Thr Thr His Val Val Pro Gly Val
Thr 580 585 590 Thr
Thr Thr Pro Thr Thr Ile Thr Ala Pro Pro Thr Gly Gln Thr Thr 595
600 605 Thr Thr Leu Val Pro Ala
Leu Pro Pro Thr Thr Thr Thr Met Ser Gly 610 615
620 His Arg Gly Ile Asn Gly Thr Val Thr Ala Thr
Ser Lys Pro Pro Ala 625 630 635
640 Val Thr Ala Gly Ser Ala Lys Val Gly Leu Val Ser Gly Ala Thr Ala
645 650 655 Ile Val
Ala Gly Val Met Ala Val Leu Met Ala Leu 660
665 149551PRTNeurospora crassa 149Met Leu Pro Val Pro Leu Thr
Thr Leu Ser Leu Tyr Val Val Ala Leu 1 5
10 15 Leu Ser Pro Pro Ala Ala Ala Gly Val Leu Ala
Ser Ala Thr Thr Lys 20 25
30 Leu Pro Ile Lys Leu Pro Ile Ser Pro Ala Gln Gly His Arg Ser
Ser 35 40 45 Thr
Ala Ala Ser Pro Ser Leu Thr Ser Arg Ser Ser Ser Ser Gly Asn 50
55 60 Gly Phe Ile Arg Ala Ser
Val His Ala Ala His Gly Ala Pro Pro Lys 65 70
75 80 Leu Arg Arg Arg Gln Glu Asp Glu Gly Leu Lys
Asn Gln Asn Leu Gly 85 90
95 Thr Thr Tyr Thr Ile Asp Ile Asp Ile Gly Thr Pro Pro Gln Thr Val
100 105 110 Thr Leu
Ile Leu Asp Thr Gly Ser Pro Asp Leu Trp Val Asn Pro Gln 115
120 125 Cys Glu Thr Ser Gly Gln Glu
Lys Tyr Cys Asn Ser Phe Arg Gln Phe 130 135
140 Asp Tyr Thr Lys Ser Lys Thr Ile Gln Asp Thr Gly
Ala Ala Asp Ile 145 150 155
160 Leu Lys Tyr Gly Lys Gly Asn Val Thr Ile Glu Tyr Val Thr Asp Asp
165 170 175 Val Ile Ile
Gly Ser Ala Lys Ile Lys Ser Gln Ile Leu Gly Ile Gly 180
185 190 Phe Glu Ser Ile Asp Ile Pro Leu
Gly Ile Leu Gly Leu Ser Pro Ser 195 200
205 Val Ser Pro Asp Gly Thr Ser Pro Tyr Pro Tyr Leu Leu
Asp Ser Met 210 215 220
Ala Ser Gln Gly Ile Ile Ser Ser Arg Ala Phe Ser Leu Asp Leu Arg 225
230 235 240 Ser Ile Asp Asn
Pro Ser Gly Ala Ile Ile Phe Gly Gly Val Asp Leu 245
250 255 Gly Lys Phe Ser Gly Ser Leu Ala Lys
Leu Pro Met Leu Asp Pro Ser 260 265
270 Gln Thr Pro Ala Gly Val Asp Arg Tyr Trp Ile Val Leu Ser
Gly Val 275 280 285
Gly Met Thr Tyr Pro Asp Gly Glu Glu Val Glu Ser Glu Glu Ile Gly 290
295 300 Val Pro Val Phe Leu
Asp Ser Gly Gly Thr Leu Ser Arg Leu Pro Glu 305 310
315 320 Thr Ile Phe Gln Ala Ile Gly Asp Ser Phe
Pro Gly Ser Gln Tyr Asp 325 330
335 Pro Glu Ser Gly Phe Tyr Ile Val Asp Cys Ala Val Ala Glu Gln
Ala 340 345 350 Gly
Ser Val Asp Phe Ile Phe Gly Ser Ser Gly Ser Arg Ser Ser Lys 355
360 365 Lys Ile Arg Val Pro Tyr
Gly Asp Phe Val Trp Glu Val Gln Thr Gly 370 375
380 Val Cys Val Val Gly Val Leu Pro Thr Asp Asp
Glu Pro Val Phe Gly 385 390 395
400 Asp Ser Phe Leu Arg Ala Ala Tyr Val Val Phe Asp Gln Asp Asn Arg
405 410 415 Asn Leu
His Leu Ala Gln Ala Ala Asn Cys Gly Glu Gln Ile Val Glu 420
425 430 Ile Gly Ser Gly Gln Asp Ala
Val Pro Ser Ser Thr Gly Lys Cys Lys 435 440
445 Asp Gly Ser Ala Gly Ser Thr Lys Thr Ala Gly Gly
Gly Gly Leu Asp 450 455 460
Val Thr Ala Thr Arg Ala Pro Thr Arg Thr Ala Gly Gly Ser Gly Pro 465
470 475 480 Ala Val Thr
Asn Ser Asp Phe Gly Pro Gly Pro Ala Gly Thr Arg Val 485
490 495 Ser Thr Gly Gly Ile Gly Leu Pro
Thr Gly Thr Gly Gly Gly Gly Gly 500 505
510 Ser Gly Asp Gly Asn Gly Asn Asn Asp Asp Asp Asp Ser
Ala Ala Ser 515 520 525
Gly Leu Asp Val Gly Val Thr Ala Ala Ala Val Leu Ala Gly Leu Asn 530
535 540 Met Leu Ile Val
Trp Leu Leu 545 550 150529PRTNeurospora crassa 150Met
Lys Ser Thr Leu Ala Thr Leu Leu Ala Leu Ala Ser Val Ala Val 1
5 10 15 Ala Glu Asn Gly Val Val
Asn Phe Pro Leu Asn Arg Gly Val Pro His 20
25 30 Phe Arg Val Gly Asn Val Arg Gln Asn Val
Lys Arg Asp Thr Tyr Ser 35 40
45 Gln Ala Leu Ile Asn Asn Ile Thr Gly Gly Ala Tyr Tyr Ala
Glu Val 50 55 60
Thr Val Gly Thr Pro Gly Gln Lys Val Ser Val Val Leu Asp Thr Gly 65
70 75 80 Ser Ser Asp Leu Trp
Val Val Ser Tyr Lys Ala Asp Leu Cys Thr Asp 85
90 95 Pro Ser Ile Gln Arg Gln Trp Gly Asp Ser
Cys Asp Lys Thr Tyr Asn 100 105
110 Pro Thr Lys Ser Ser Ser Tyr Lys Val Leu Glu Glu Asp Ser Phe
Glu 115 120 125 Ile
Arg Tyr Leu Asp Asn Ser Thr Ala Ala Gly Asp Tyr Ile Thr Asp 130
135 140 Asp Leu Asn Ile Gly Gly
Ala Thr Ile Lys Ser Leu Gln Met Gly Tyr 145 150
155 160 Ala Thr Lys Thr Val Arg Gly Ala Gly Ile Leu
Gly Val Gly Tyr Ser 165 170
175 Ser Asn Val Ala Ser Gln Gln Arg Tyr Pro Asn Leu Ile Asp Gln Phe
180 185 190 Val Ala
Gln Lys Leu Ile Thr Thr Lys Ala Tyr Ser Leu Tyr Leu Asn 195
200 205 Asp Arg Arg Ser Asp Thr Gly
Ser Ile Leu Phe Gly Gly Ile Asp Lys 210 215
220 Asp Lys Phe Ile Gly Asp Leu Ser Ile Leu Pro Ile
Tyr Leu Ala Lys 225 230 235
240 Gly Gln Ala Glu Pro Ile His Phe Glu Val Glu Met Gln Ser Val Ser
245 250 255 Leu Ala Leu
Thr Lys Asn Gly Lys Thr Thr Lys Ile Ile Ser Thr Asp 260
265 270 Pro Ser Leu Ser Gln Thr Ser Thr
Ile Ala Ile Leu Asp Ser Gly Thr 275 280
285 Thr Leu Ser Tyr Leu Pro Ser Lys Ile Thr Asp Gln Ile
His Thr Lys 290 295 300
Leu Ser Val Tyr Val Asp Glu Ile Trp Thr Gly Leu Thr Phe Ile Asp 305
310 315 320 Cys Gln Tyr Leu
Thr Ser Asn Pro Asp Leu Arg Leu Ser Phe Thr Phe 325
330 335 Gly Ala Asn Ala Thr Ile Ser Val Pro
Val Trp Glu Leu Val Leu Asp 340 345
350 Leu Leu Gly Glu Ser Gln Ser Glu Leu Pro Phe Lys Met Pro
Phe Lys 355 360 365
Asn Ala Cys Ile Phe Gly Ile Gln Ser Thr Ala Gly Phe Gln Glu Asp 370
375 380 Asn Phe Asp Glu Asp
Trp Ala Leu Leu Gly Glu Thr Phe Leu Arg Ser 385 390
395 400 Ala Tyr Val Val Tyr Asp Leu Thr His His
Gln Ile Gly Ile Ala Gln 405 410
415 Ala Asn Leu Asn Ser Thr Thr Thr Asp Ile Val Glu Leu Ser Gly
Ala 420 425 430 Asp
Gly Gly Leu Pro Thr Gly Leu Thr Gly Val Lys Glu Gln Gln Thr 435
440 445 Ser Asn Asp Pro Ser Gly
Asn Ala Gly Ser Gly Ser Gly Ser Ser Thr 450 455
460 Asp Lys Asp Gly Ala Lys Glu Thr Glu Thr Val
Thr Ala Gly Ser Thr 465 470 475
480 Ala Ala Thr Gly Thr Ala Ala Ser Gly Ala Lys Glu Thr Asp Ser Ala
485 490 495 Ala Ala
Gly Leu Ser Ala Arg Gly Gly Ala Val Gly Ala Leu Ala Val 500
505 510 Ala Ser Leu Thr Gly Phe Leu
Ala Leu Val Gly Gly Ala Val Val Ala 515 520
525 Leu 151566PRTMyceliophthora thermophila 151Met
Lys Pro Ser Ser Ala Ile Leu Leu Ala Leu Ala Pro Gly Ser Ser 1
5 10 15 Ser Lys Asn Val Val Glu
Phe Ser Val Ser Arg Gly Leu Pro Gly Asn 20
25 30 Arg Thr Pro Leu Ser Phe Pro Pro Leu Thr
Arg Arg Glu Thr Tyr Ser 35 40
45 Glu Arg Leu Ile Asn Asn Ile Ala Gly Gly Gly Tyr Tyr Val
Gln Val 50 55 60
Gln Val Gly Thr Pro Pro Gln Asn Leu Thr Met Leu Leu Asp Thr Gly 65
70 75 80 Ser Ser Asp Ala Trp
Val Leu Ser His Glu Ala Asp Leu Cys Ile Ser 85
90 95 Pro Ala Leu Gln Asp Phe Tyr Gly Met Pro
Cys Thr Asp Thr Tyr Asp 100 105
110 Pro Ser Lys Ser Ser Ser Lys Lys Met Val Glu Glu Gly Gly Phe
Lys 115 120 125 Ile
Thr Tyr Leu Asp Gly Gly Thr Ala Ser Gly Asp Tyr Ile Thr Asp 130
135 140 His Phe Thr Ile Gly Gly
Val Thr Val Gln Ser Leu Gln Met Ala Cys 145 150
155 160 Val Thr Lys Ala Val Arg Gly Thr Gly Ile Leu
Gly Leu Gly Phe Ser 165 170
175 Ile Ser Glu Arg Ala Ser Thr Lys Tyr Pro Asn Ile Ile Asp Glu Met
180 185 190 Tyr Ser
Gln Gly Leu Ile Lys Ser Lys Ala Phe Ser Leu Tyr Leu Asn 195
200 205 Asp Arg Arg Ala Asp Ser Gly
Thr Leu Leu Phe Gly Gly Ile Asp Thr 210 215
220 Asp Lys Phe Ile Gly Pro Leu Gly Val Leu Pro Leu
His Lys Pro Pro 225 230 235
240 Gly Asp Arg Asp Tyr Ser Ser Phe Glu Val Asn Phe Thr Ser Val Ser
245 250 255 Leu Thr Tyr
Thr Asn Gly Ser Arg His Thr Ile Pro Thr Ala Ile Leu 260
265 270 Asn His Pro Ala Pro Ala Val Leu
Asp Ser Gly Thr Thr Leu Ser Tyr 275 280
285 Leu Pro Asp Glu Leu Ala Asp Pro Ile Asn Thr Ala Leu
Asp Thr Phe 290 295 300
Tyr Asp Asp Arg Leu Gln Met Thr Leu Ile Asp Cys Ser His Pro Leu 305
310 315 320 Leu Arg Thr Asp
Pro Asp Phe His Leu Ala Phe Thr Phe Thr Pro Thr 325
330 335 Thr Ser Ile Thr Val Pro Leu Gly Asp
Leu Val Leu Asp Ile Leu Pro 340 345
350 Pro Thr Tyr Pro Gln Ser Asn Ser Asn Asn Asn Asn Glu Val
Glu Asp 355 360 365
Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Val Pro Pro 370
375 380 Ala Thr Glu Arg Arg
Trp Cys Val Phe Gly Ile Gln Ser Thr Thr Arg 385 390
395 400 Phe Ala Ala Ser Ser Gly Gln Ser Glu Ala
Asn Phe Thr Leu Leu Gly 405 410
415 Asp Thr Phe Leu Arg Ser Ala Tyr Val Val Tyr Asp Leu Ser His
Tyr 420 425 430 Gln
Ile Gly Leu Ala Gln Ala Asn Leu Asn Ser Ser Ser Ser Ser Thr 435
440 445 Asn Thr Asn Thr Ile Val
Glu Leu Thr Ala Asp Asn His Asp Asp Gly 450 455
460 Ala Ser Glu Arg Gly Glu Gly Ala Gly Ala Gly
Ala Asp Ala Gly Thr 465 470 475
480 Arg Thr Val Ile Ala Gly Gly Leu Pro Ser Gly Leu Met Gly Val Glu
485 490 495 Ala Gln
Gln Thr Thr Phe Thr Pro Thr Ala Thr Ala Asn Gly His Pro 500
505 510 Gly Tyr Gly Gly Gly Pro Gly
Gly Ser Thr Arg Pro Gly Ser Glu Arg 515 520
525 Asn Ala Ala Ala Gly Gly Phe Thr Ala Val Arg Thr
Gly Leu Leu Gly 530 535 540
Glu Leu Val Gly Val Ala Ala Val Thr Ala Leu Phe Ile Leu Leu Gly 545
550 555 560 Gly Ala Leu
Ile Ala Val 565 152897PRTMyceliophthora thermophila
152Met Val Arg Leu Asp Trp Ala Ala Val Leu Leu Ala Ala Thr Ala Val 1
5 10 15 Ala Lys Ala Val
Thr Pro His Thr Pro Ser Phe Val Pro Gly Ala Tyr 20
25 30 Ile Val Glu Tyr Glu Glu Asp Gln Asp
Ser His Ala Phe Val Asn Lys 35 40
45 Leu Gly Gly Lys Ala Ser Leu Arg Lys Asp Leu Arg Phe Lys
Leu Phe 50 55 60
Lys Gly Ala Ser Ile Gln Phe Lys Asp Thr Glu Thr Ala Asp Gln Met 65
70 75 80 Val Ala Lys Val Ala
Glu Met Pro Lys Val Lys Ala Val Tyr Pro Val 85
90 95 Arg Arg Tyr Pro Val Pro Asn His Val Val
His Ser Thr Gly Asn Val 100 105
110 Ala Asp Glu Val Leu Val Lys Arg Gln Ala Ala Gly Asn Asp Thr
Phe 115 120 125 Ser
Thr His Leu Met Thr Gln Val Asn Lys Phe Arg Asp Ala Gly Ile 130
135 140 Thr Gly Lys Gly Ile Lys
Ile Ala Val Ile Asp Thr Gly Ile Asp Tyr 145 150
155 160 Leu His Glu Ala Leu Gly Gly Cys Phe Gly Pro
Asp Cys Leu Val Ser 165 170
175 Tyr Gly Thr Asp Leu Val Gly Asp Asp Phe Asn Gly Ser Asn Thr Pro
180 185 190 Lys Pro
Asp Pro Asp Pro Ile Asp Asn Cys Gln Gly His Gly Thr His 195
200 205 Val Ala Gly Ile Ile Ala Ala
Gln Thr Asn Asn Pro Phe Gly Ile Ile 210 215
220 Gly Ala Ala Thr Asp Val Thr Leu Gly Ala Tyr Arg
Val Phe Gly Cys 225 230 235
240 Asn Gly Asp Thr Pro Asn Asp Val Leu Ile Ala Ala Tyr Asn Met Ala
245 250 255 Tyr Glu Ala
Gly Ser Asp Ile Ile Thr Ala Ser Ile Gly Gly Pro Ser 260
265 270 Gly Trp Ser Glu Asp Pro Trp Ala
Ala Val Val Thr Arg Ile Val Glu 275 280
285 Asn Gly Val Pro Cys Val Val Ser Ala Gly Asn Asp Gly
Asp Ala Gly 290 295 300
Ile Phe Tyr Ala Ser Thr Ala Ala Asn Gly Lys Lys Val Thr Ala Ile 305
310 315 320 Ala Ser Val Asp
Asn Ile Val Thr Pro Ala Leu Leu Ser Asn Ala Ser 325
330 335 Tyr Thr Leu Asn Gly Thr Asp Asp Phe
Phe Gly Phe Thr Ala Gly Asp 340 345
350 Pro Gly Ser Trp Asp Asp Val Asn Leu Pro Leu Trp Ala Val
Ser Phe 355 360 365
Asp Thr Thr Asp Pro Ala Asn Gly Cys Asn Pro Tyr Pro Asp Ser Thr 370
375 380 Pro Asp Leu Ser Gly
Tyr Ile Val Leu Ile Arg Arg Gly Thr Cys Thr 385 390
395 400 Phe Val Glu Lys Ala Ser Tyr Ala Ala Ala
Lys Gly Ala Lys Tyr Val 405 410
415 Met Phe Tyr Asn Asn Val Gln Gln Gly Thr Val Thr Val Ser Ala
Ala 420 425 430 Glu
Ala Lys Gly Ile Glu Gly Val Ala Met Val Thr Ala Gln Gln Gly 435
440 445 Glu Ala Trp Val Arg Ala
Leu Glu Ala Gly Ser Glu Val Val Leu His 450 455
460 Met Lys Asp Pro Leu Lys Ala Gly Lys Phe Leu
Thr Thr Thr Pro Asn 465 470 475
480 Thr Ala Thr Gly Gly Phe Met Ser Asp Tyr Thr Ser Trp Gly Pro Thr
485 490 495 Trp Glu
Val Glu Val Lys Pro Gln Phe Gly Thr Pro Gly Gly Ser Ile 500
505 510 Leu Ser Thr Tyr Pro Arg Ala
Leu Gly Ser Tyr Ala Val Leu Ser Gly 515 520
525 Thr Ser Met Ala Cys Pro Leu Ala Ala Ala Ile Tyr
Ala Leu Leu Ile 530 535 540
Asn Thr Arg Gly Thr Lys Asp Pro Lys Thr Leu Glu Asn Leu Ile Ser 545
550 555 560 Ser Thr Ala
Arg Pro Asn Leu Phe Arg Leu Asn Gly Glu Ser Leu Pro 565
570 575 Leu Leu Ala Pro Val Pro Gln Gln
Gly Gly Gly Ile Val Gln Ala Trp 580 585
590 Asp Ala Ala Gln Ala Thr Thr Leu Leu Ser Val Ser Ser
Leu Ser Phe 595 600 605
Asn Asp Thr Asp His Phe Lys Pro Val Gln Thr Phe Thr Ile Thr Asn 610
615 620 Thr Gly Lys Lys
Ala Val Thr Tyr Ser Leu Ser Asn Val Gly Ala Ala 625 630
635 640 Thr Ala Tyr Thr Phe Ala Asp Ala Lys
Ser Ile Glu Pro Ala Pro Phe 645 650
655 Pro Asn Glu Leu Thr Ala Asp Phe Ala Ser Leu Thr Phe Val
Pro Lys 660 665 670
Arg Leu Thr Ile Pro Ala Gly Lys Arg Gln Thr Val Thr Val Ile Ala
675 680 685 Lys Pro Ser Glu
Gly Val Asp Ala Lys Arg Leu Pro Val Tyr Ser Gly 690
695 700 Tyr Ile Ala Ile Asn Gly Ser Asp
Ser Ser Ala Leu Ser Leu Pro Tyr 705 710
715 720 Leu Gly Val Val Gly Ser Leu His Ser Ala Val Val
Leu Asp Ser Asn 725 730
735 Gly Ala Arg Ile Ser Leu Ala Ser Asp Asp Thr Asn Lys Pro Leu Pro
740 745 750 Ala Asn Thr
Ser Phe Val Leu Pro Pro Ala Gly Phe Pro Asn Asp Thr 755
760 765 Ser Tyr Ala Asn Ser Thr Asp Leu
Pro Lys Leu Val Val Asp Leu Ala 770 775
780 Met Gly Ser Ala Leu Leu Arg Ala Asp Val Val Pro Leu
Ser Gly Gly 785 790 795
800 Ala Ala Thr Ala Thr Ala Arg Leu Thr Arg Thr Val Phe Gly Thr Arg
805 810 815 Thr Ile Gly Gln
Pro Tyr Gly Leu Pro Ala Arg Tyr Asn Pro Arg Gly 820
825 830 Thr Phe Glu Tyr Ala Trp Asp Gly Arg
Leu Asp Asp Gly Ser Tyr Ala 835 840
845 Pro Ala Gly Arg Tyr Arg Phe Ala Val Lys Ala Leu Arg Ile
Phe Gly 850 855 860
Asp Ala Lys Arg Ala Arg Glu Tyr Asp Ala Ala Glu Thr Val Glu Phe 865
870 875 880 Asn Ile Glu Tyr Leu
Pro Gly Pro Ser Ala Lys Phe Arg Arg Arg Leu 885
890 895 Phe 153876PRTNeurospora crassa 153Met
Val Arg Leu Gly Leu Ala Thr Thr Leu Leu Ala Ala Ala Ser Phe 1
5 10 15 Ala Gln Ala Ala His Gln
Lys Ala Pro Ala Val Val Pro Gly Ala Tyr 20
25 30 Ile Val Glu Tyr Glu Asp Ser His Asp Pro
Thr Ser Ile Leu Ala Ser 35 40
45 Ile Lys Gly Asp Ala Thr Ile Arg Lys Asp Ile Arg His Glu
Leu Phe 50 55 60
Lys Gly Ala Ser Phe Gln Phe Lys Asp Leu Asn Lys Ala Asp Asp Leu 65
70 75 80 Ala Ser Lys Val Ala
Ala Met Ser Gly Val Lys Ala Leu Tyr Pro Val 85
90 95 Arg Arg Tyr Ser Ile Pro Glu His Thr Val
His Ser Thr Gly Ser Ala 100 105
110 Val Gln Glu Val Val Ala Lys Arg Asp Thr Gly Asn Asp Thr Phe
Ser 115 120 125 Pro
His Leu Met Thr Gln Val Asn Lys Phe Arg Asp Ser Gly Ile Thr 130
135 140 Gly Lys Gly Ile Lys Ile
Ala Val Ile Asp Thr Gly Val Asp Tyr Leu 145 150
155 160 His Pro Ala Leu Gly Gly Cys Phe Gly Pro Gly
Cys Leu Val Ser Tyr 165 170
175 Gly Thr Asp Leu Val Gly Asp Asp Phe Asn Gly Ser Asn Thr Pro Val
180 185 190 Pro Asp
Ser Asp Pro Met Asp Thr Cys Asn Gly His Gly Ser His Val 195
200 205 Leu Gly Leu Leu Ser Ala Asn
Thr Asn Asn Pro Tyr Gly Ile Ile Gly 210 215
220 Ala Ala Pro Asp Val Thr Leu Gly Ala Tyr Arg Val
Phe Gly Cys Ser 225 230 235
240 Gly Asp Val Gly Asn Asp Ile Leu Ile Glu Ala Tyr Leu Lys Ala Tyr
245 250 255 Asp Asp Gly
Ser Asp Ile Ile Thr Ala Ser Ile Gly Gly Ala Ser Gly 260
265 270 Trp Pro Glu Asp Ser Trp Ala Ala
Val Val Ser Arg Ile Val Glu Lys 275 280
285 Gly Val Pro Cys Leu Val Ser Ala Gly Asn Asp Gly Ala
Thr Gly Ile 290 295 300
Phe Tyr Ala Ser Thr Ala Ala Asn Gly Lys Arg Val Thr Ala Val Ala 305
310 315 320 Ser Val Asp Asn
Ile Leu Ala Pro Ala Leu Leu Ser Glu Ala Ser Tyr 325
330 335 Ser Val Ala Asn Gly Ser Leu Ser Thr
Phe Gly Phe Thr Ala Gly Ser 340 345
350 Pro Ser Ala Trp Ala Asn Val Ser Leu Pro Val Trp Ser Val
Asn Phe 355 360 365
Asn Thr Ala Asp Ala Ala Asn Gly Cys Glu Ala Phe Pro Asp Asp Thr 370
375 380 Pro Asp Leu Ser Lys
Tyr Ile Val Leu Ile Arg Arg Gly Thr Cys Thr 385 390
395 400 Phe Val Gln Lys Ala Gln Asn Ala Ala Ala
Lys Gly Ala Lys Tyr Ile 405 410
415 Ile Tyr Tyr Asn Asn Ala Ser Gly Ser Thr Lys Val Asp Val Ser
Ala 420 425 430 Val
Ala Asp Val Lys Ala Ala Ala Met Val Thr Ser Glu Thr Gly Ala 435
440 445 Ala Trp Ile Lys Ala Leu
Gln Ala Gly Thr Gln Val Thr Val Asn Met 450 455
460 Ala Asp Pro Glu Thr Ala Pro Lys Asn Leu Asn
Asn Phe Pro Asn Thr 465 470 475
480 Ala Thr Pro Gly Phe Leu Ser Thr Tyr Thr Ser Trp Gly Pro Thr Tyr
485 490 495 Glu Val
Asp Val Lys Pro Gln Ile Ser Ser Pro Gly Gly Met Ile Leu 500
505 510 Ser Thr Tyr Pro Arg Ala Leu
Gly Ser Tyr Ala Val Leu Ser Gly Thr 515 520
525 Ser Met Ala Cys Pro Leu Ala Ala Ala Thr Trp Ala
Leu Val Met Gln 530 535 540
Lys Arg Gly Thr Lys Asp Pro Lys Val Leu Glu Asn Leu Phe Ser Ala 545
550 555 560 Thr Ala His
Pro Asn Leu Phe Asn Asp Gly Thr Lys Thr Tyr Pro Met 565
570 575 Leu Ala Pro Val Ala Gln Gln Gly
Ala Gly Leu Ile Gln Ala Trp Asp 580 585
590 Ala Ala Asn Ala Asn Ala Leu Leu Ser Val Ser Ser Ile
Ser Phe Asn 595 600 605
Asp Thr Glu His Phe Lys Pro Leu Gln Ser Phe Glu Val Thr Asn Thr 610
615 620 Gly Lys Lys Ala
Val Thr Tyr Gln Leu Gly His Thr Ser Ala Ala Thr 625 630
635 640 Ala Tyr Thr Phe Ala Asn Asp Thr Ser
Ile Gly Pro Ala Ala Phe Pro 645 650
655 Asn Glu Leu Val Asp Ala Lys Ala Thr Leu Val Leu Thr Pro
Ala Lys 660 665 670
Leu Thr Leu Asn Pro Gly Gln Lys Lys Thr Val Thr Val Leu Ala Ile
675 680 685 Pro Pro Leu Gly
Leu Asp Ala Lys Arg Leu Pro Val Tyr Ser Gly Tyr 690
695 700 Ile Thr Leu Asn Gly Thr Asp Ser
Thr Gly Tyr Ser Leu Pro Tyr Gln 705 710
715 720 Gly Val Val Gly Ser Met Arg Ser Val Thr Val Leu
Asp Lys Gln Asn 725 730
735 Ser Tyr Leu Ser Gln Ser Ser Asp Ala Thr Tyr Ala Pro Val Ala Ala
740 745 750 Gly Thr Thr
Phe Thr Leu Pro Pro Ala Gly Lys Ala Asn Asp Thr Leu 755
760 765 Tyr Ala Thr Thr Val Tyr Pro Thr
Ile Val Leu Thr Leu Ser Met Gly 770 775
780 Ser Ala Glu Val His Ala Asp Val Val Asn Ser Lys Gly
Lys Thr Ile 785 790 795
800 Gly Gln Val Leu Thr Phe Pro Ala Arg Trp Asn Pro Arg Gly Thr Phe
805 810 815 Glu Trp Asn Trp
Asp Gly Ala Leu Ser Asp Gly Thr Tyr Ala Pro Ala 820
825 830 Asp Thr Tyr Lys Ile Thr Leu Lys Ala
Leu Lys Ile Tyr Gly Asn Ser 835 840
845 Lys Trp Pro Leu Asp Trp Glu Thr Gln Thr Thr Glu Pro Phe
Thr Ile 850 855 860
Lys Tyr Ala Ala Lys Ser Lys Arg Ala Phe Thr Ala 865 870
875 154534PRTMyceliophthora thermophila 154Met Arg Gly
Leu Val Ala Phe Ser Leu Ala Ala Cys Val Ser Ala Ala 1 5
10 15 Pro Ser Phe Lys Thr Glu Thr Ile
Asn Gly Glu His Ala Pro Ile Leu 20 25
30 Ser Ser Ser Asn Ala Glu Val Val Pro Asn Ser Tyr Ile
Ile Lys Phe 35 40 45
Lys Lys His Val Asp Glu Ser Ser Ala Ser Ala His His Ala Trp Ile 50
55 60 Gln Asp Ile His
Thr Ser Arg Glu Lys Val Arg Gln Asp Leu Lys Lys 65 70
75 80 Arg Gly Gln Val Pro Leu Leu Asp Asp
Val Phe His Gly Leu Lys His 85 90
95 Thr Tyr Lys Ile Gly Gln Glu Phe Leu Gly Tyr Ser Gly His
Phe Asp 100 105 110
Asp Glu Thr Ile Glu Gln Val Arg Arg His Pro Asp Val Glu Tyr Ile
115 120 125 Glu Arg Asp Ser
Ile Val His Thr Met Arg Val Thr Glu Glu Thr Cys 130
135 140 Asp Gly Glu Leu Glu Lys Ala Ala
Pro Trp Gly Leu Ala Arg Ile Ser 145 150
155 160 His Arg Asp Thr Leu Gly Phe Ser Thr Phe Asn Lys
Tyr Leu Tyr Ala 165 170
175 Ala Glu Gly Gly Glu Gly Val Asp Ala Tyr Val Ile Asp Thr Gly Thr
180 185 190 Asn Ile Glu
His Val Asp Phe Glu Gly Arg Ala Lys Trp Gly Lys Thr 195
200 205 Ile Pro Ala Gly Asp Ala Asp Val
Asp Gly Asn Gly His Gly Thr His 210 215
220 Cys Ser Gly Thr Ile Ala Gly Lys Lys Tyr Gly Val Ala
Lys Lys Ala 225 230 235
240 Asn Val Tyr Ala Val Lys Val Leu Arg Ser Asn Gly Ser Gly Thr Met
245 250 255 Ala Asp Val Val
Ala Gly Val Glu Trp Ala Ala Lys Ser His Leu Glu 260
265 270 Gln Val Gln Ala Ala Lys Asp Gly Lys
Arg Lys Gly Phe Lys Gly Ser 275 280
285 Val Ala Asn Met Ser Leu Gly Gly Gly Lys Thr Arg Ala Leu
Asp Asp 290 295 300
Thr Val Asn Ala Ala Val Ser Val Gly Ile His Phe Ala Val Ala Ala 305
310 315 320 Gly Asn Asp Asn Ala
Asp Ala Cys Asn Tyr Ser Pro Ala Ala Ala Glu 325
330 335 Lys Ala Val Thr Val Gly Ala Ser Ala Ile
Asp Asp Ser Arg Ala Tyr 340 345
350 Phe Ser Asn Tyr Gly Lys Cys Thr Asp Ile Phe Ala Pro Gly Leu
Ser 355 360 365 Ile
Leu Ser Thr Trp Ile Gly Ser Lys Tyr Ala Thr Asn Thr Ile Ser 370
375 380 Gly Thr Ser Met Ala Ser
Pro His Ile Ala Gly Leu Leu Ala Tyr Tyr 385 390
395 400 Leu Ser Leu Gln Pro Ala Thr Asp Ser Glu Tyr
Ser Val Ala Pro Ile 405 410
415 Thr Pro Glu Lys Met Lys Ser Asn Leu Leu Lys Ile Ala Thr Gln Asp
420 425 430 Ala Leu
Thr Asp Ile Pro Asp Glu Thr Pro Asn Leu Leu Ala Trp Asn 435
440 445 Gly Gly Gly Cys Asn Asn Tyr
Thr Ala Ile Val Glu Ala Gly Gly Tyr 450 455
460 Lys Ala Lys Lys Lys Thr Thr Thr Asp Lys Val Asp
Ile Gly Ala Ser 465 470 475
480 Val Ser Glu Leu Glu Lys Leu Ile Glu His Asp Phe Glu Val Ile Ser
485 490 495 Gly Lys Val
Val Lys Gly Val Ser Ser Phe Ala Asp Lys Ala Glu Lys 500
505 510 Phe Ser Glu Lys Ile His Glu Leu
Val Asp Glu Glu Leu Lys Glu Phe 515 520
525 Leu Glu Asp Ile Ala Ala 530
155396PRTNeurospora crassa 155Met Lys Leu Ser Ala Val Leu Ala Leu Leu Pro
Leu Ala Met Ala Ala 1 5 10
15 Pro Ser Ala Pro Ile Asp Lys Arg Ala Pro Ile Leu Glu Ala Arg Ala
20 25 30 Gly Thr
Gln Ala Val Pro Gly Lys Tyr Ile Val Lys Leu Arg Glu Thr 35
40 45 Ala Ser Asp Asp Asp Leu Asp
Lys Ala Val Lys Lys Leu Gly Asn Ser 50 55
60 Lys Ala Asp His Val Tyr Lys His Ala Phe Arg Gly
Phe Ala Gly Arg 65 70 75
80 Ile Asp Asp Lys Thr Leu Asp Asp Ile Arg Ser Leu Pro Glu Val Glu
85 90 95 Tyr Val Glu
Gln Glu Ala Val Phe Thr Ile Asn Thr Tyr Thr Ser Gln 100
105 110 Ser Ser Val Pro Ser Trp Gly Leu
Ala Arg Leu Ser Ser Lys Thr Thr 115 120
125 Gly Lys Thr Thr Tyr Val Tyr Asp Ser Ser Ala Gly Ala
Gly Thr Cys 130 135 140
Ala Tyr Ile Ile Asp Thr Gly Ile Asn Thr Ala His Ser Asp Phe Gly 145
150 155 160 Gly Arg Ala Thr
Trp Leu Ala Asn Tyr Ala Gly Asp Gly Ile Asn Ser 165
170 175 Asp Gly Asn Gly His Gly Thr His Val
Ala Gly Thr Val Gly Gly Thr 180 185
190 Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Tyr Ala Val Lys
Val Leu 195 200 205
Asp Ser Asn Gly Ser Gly Ser Asn Ser Gly Val Ile Ala Gly Met Asn 210
215 220 Phe Val Ala Gln Asp
Ala Gln Ser Arg Asn Cys Pro Asn Gly Thr Val 225 230
235 240 Ala Asn Met Ser Leu Gly Gly Gly Tyr Ser
Ala Ser Thr Asn Ser Ala 245 250
255 Ala Ala Ala Met Val Arg Ala Gly Val Phe Leu Ala Val Ala Ala
Gly 260 265 270 Asn
Asp Gly Ala Asn Ala Ala Asn Tyr Ser Pro Ala Ser Glu Pro Thr 275
280 285 Val Cys Thr Val Gly Ala
Thr Thr Ser Ala Asp Ala Ile Ala Tyr Tyr 290 295
300 Ser Asn Tyr Gly Thr Ile Val Asp Ile Phe Ala
Pro Gly Thr Ser Ile 305 310 315
320 Thr Ser Ala Trp Ile Gly Ser Thr Thr Ala Lys Asn Thr Ile Ser Gly
325 330 335 Thr Ser
Met Ala Thr Pro His Ile Thr Gly Leu Gly Ala Tyr Leu Leu 340
345 350 Thr Leu Leu Gly Lys Lys Ser
Pro Ala Ala Leu Cys Ser Tyr Ile Ala 355 360
365 Ser Thr Ala Asn Ser Gly Val Ile Ser Gly Ile Pro
Arg Gly Thr Val 370 375 380
Asn Lys Leu Ala Phe Asn Gly Asn Pro Ser Ala Tyr 385
390 395 156392PRTMyceliophthora thermophila 156Met
His Phe Ser Thr Ala Leu Leu Ala Phe Leu Pro Ala Ala Leu Ala 1
5 10 15 Ala Pro Thr Ala Glu Thr
Leu Asp Lys Arg Ala Pro Ile Leu Thr Ala 20
25 30 Arg Ala Gly Gln Val Val Pro Gly Lys Tyr
Ile Ile Lys Leu Arg Asp 35 40
45 Gly Ala Ser Asp Asp Val Leu Glu Ala Ala Ile Gly Lys Leu
Arg Ser 50 55 60
Lys Ala Asp His Val Tyr Arg Gly Lys Phe Arg Gly Phe Ala Gly Lys 65
70 75 80 Leu Glu Asp Asp Val
Leu Asp Ala Ile Arg Leu Leu Pro Glu Val Glu 85
90 95 Tyr Val Glu Glu Glu Ala Ile Phe Thr Ile
Asn Ala Tyr Thr Ser Gln 100 105
110 Ser Asn Ala Pro Trp Gly Leu Ala Arg Leu Ser Ser Lys Thr Ala
Gly 115 120 125 Ser
Thr Thr Tyr Thr Tyr Asp Thr Ser Ala Gly Glu Gly Thr Cys Ala 130
135 140 Tyr Val Ile Asp Thr Gly
Ile Tyr Thr Ser His Ser Asp Phe Gly Gly 145 150
155 160 Arg Ala Thr Phe Ala Ala Asn Phe Val Asp Ser
Ser Asn Thr Asp Gly 165 170
175 Asn Gly His Gly Thr His Val Ala Gly Thr Ile Gly Gly Thr Thr Tyr
180 185 190 Gly Val
Ala Lys Lys Thr Lys Leu Tyr Ala Val Lys Val Leu Gly Ser 195
200 205 Asp Gly Ser Gly Thr Thr Ser
Gly Val Ile Ala Gly Ile Asn Phe Val 210 215
220 Ala Asp Asp Ala Pro Lys Arg Ser Cys Pro Lys Gly
Val Val Ala Asn 225 230 235
240 Met Ser Leu Gly Gly Ser Tyr Ser Ala Ser Ile Asn Asn Ala Ala Ala
245 250 255 Ala Leu Val
Arg Ser Gly Val Phe Leu Ala Val Ala Ala Gly Asn Glu 260
265 270 Asn Gln Asn Ala Ala Asn Ser Ser
Pro Ala Ser Glu Ala Ser Ala Cys 275 280
285 Thr Val Gly Ala Thr Asp Arg Asn Asp Ala Lys Ala Ser
Tyr Ser Asn 290 295 300
Tyr Gly Ser Val Val Asp Ile Gln Ala Pro Gly Ser Asn Ile Leu Ser 305
310 315 320 Thr Trp Ile Gly
Ser Thr Ser Ala Thr Asn Thr Ile Ser Gly Thr Ser 325
330 335 Met Ala Ser Pro His Ile Ala Gly Leu
Gly Ala Tyr Leu Leu Ala Leu 340 345
350 Glu Gly Ser Lys Thr Pro Ala Glu Leu Cys Asn Tyr Ile Lys
Ser Thr 355 360 365
Gly Asn Ala Ala Ile Thr Gly Val Pro Ser Gly Thr Thr Asn Arg Ile 370
375 380 Ala Phe Asn Gly Asn
Pro Ser Ala 385 390 157433PRTNeurospora crassa
157Met Val Arg Phe Ser Val Ala Ala Ala Phe Leu Leu Ser Ala Leu Gly 1
5 10 15 Val Thr Ala Ala
Pro Ser Gly Gly Arg His Asn His Gln Asn Thr Gln 20
25 30 Asn Thr Gly Ala Thr Ala Gly Asn Ala
Ala Gly Val Pro Val Ala Asn 35 40
45 Ser Asp Ile Ser Asn Ile Ile Pro Gly Arg Tyr Ile Val Val
Tyr Asn 50 55 60
Asn Thr Phe Gly Glu Glu Ala Ile Asn Ala His Gln Ile Lys Val Thr 65
70 75 80 Ser Leu Val Ala Lys
Arg Asn Leu Gly Lys Arg Asp Ala Lys Thr Gly 85
90 95 Arg Ile Met Ser Pro Ser Val Lys Ala Phe
Lys Met Gly Thr Trp Arg 100 105
110 Ala Met Ala Leu Asp Ala Asp Asp Asp Met Ile Asn Asp Ile Asn
Ser 115 120 125 Ala
Gln Glu Val Glu Tyr Ile Glu Ala Asp Gln Tyr Val Lys Leu Asn 130
135 140 Ala Leu Thr Ser Gln Asn
Ser Thr Thr Thr Gly Leu Ala Arg Leu Ser 145 150
155 160 His Ala Gly Pro Ser Lys Lys Ala Ala Pro Tyr
Ile Phe Asp Ser Ser 165 170
175 Ala Gly Glu Gly Ile Thr Ala Phe Val Val Asp Thr Gly Ile Arg Val
180 185 190 Thr His
Ser Glu Tyr Glu Gly Arg Ala Thr Phe Ala Ala Asn Phe Val 195
200 205 Asn Asn Val Asp Thr Asp Glu
Asn Gly His Gly Ser His Val Ala Gly 210 215
220 Thr Ile Ala Gly Ala Thr Phe Gly Val Ala Lys Lys
Ala Lys Leu Val 225 230 235
240 Ala Val Lys Val Leu Asp Gly Ser Gly Ser Gly Ser Asn Ser Gly Val
245 250 255 Leu Gln Gly
Met Gln Phe Val Ala Asp Thr Ala Thr Ser Gln Lys Leu 260
265 270 Gly Gly Lys Ala Val Leu Asn Met
Ser Leu Gly Gly Gly Lys Ser Arg 275 280
285 Ala Ile Asn Ser Ala Ile Asn Gln Ile Ala Ala Ala Gly
Val Val Pro 290 295 300
Val Val Ala Ala Gly Asn Glu Asn Gln Asp Thr Ala Asn Thr Ser Pro 305
310 315 320 Gly Ser Ala Pro
Ala Ala Ile Thr Val Gly Ala Ile Asp Gln Arg Thr 325
330 335 Asp Ala Arg Ala Ser Phe Ser Asn Phe
Gly Ala Gly Val Asp Ile Phe 340 345
350 Ala Pro Gly Val Asn Val Leu Ser Val Gly Ile Lys Ser Asp
Thr Asp 355 360 365
Thr Asp Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly 370
375 380 Leu Ala Ala Tyr Leu
Met Ala Leu Glu Gly Leu Thr Asp Val Thr Ala 385 390
395 400 Val Gly Asn Arg Ile Lys Glu Leu Ala Gln
Lys Thr Gly Ala Lys Val 405 410
415 Thr Asn Asn Val Arg Gly Thr Thr Ser Leu Ile Ala Asn Asn Gly
Asn 420 425 430 Leu
158420PRTMyceliophthora thermophila 158Met Ala Gly Arg Leu Leu Leu Cys
Leu Thr Ala Ala Leu Ser Ala Leu 1 5 10
15 Gly Val Ser Ala Ala Pro Ala Pro Asp Ala Ser Gly Arg
Pro Phe Ile 20 25 30
Gly Val Pro Val Ser Asn Pro Gly Ile Ala Asn Ala Ile Pro Asn Arg
35 40 45 Tyr Ile Val Val
Tyr Asn Asn Thr Phe Asn Asp Glu Asp Ile Asp Leu 50
55 60 His Gln Ser Asn Val Ile Lys Thr
Ile Ala Lys Arg Asn Ile Ala Lys 65 70
75 80 Arg Ser Leu Thr Gly Lys Leu Leu Ser Thr Thr Val
Asn Thr Tyr Lys 85 90
95 Ile Asn Asn Trp Arg Ala Met Ala Leu Glu Ala Asp Asp Ala Thr Ile
100 105 110 Asn Glu Ile
Phe Ala Ala Lys Glu Val Ser Tyr Ile Glu Gln Asp Ala 115
120 125 Val Ile Ser Leu Asn Val Arg Gln
Met Gln Ser Gln Ala Thr Thr Gly 130 135
140 Leu Ala Arg Ile Ser His Ala Gln Pro Gly Ala Arg Thr
Tyr Ile Phe 145 150 155
160 Asp Ser Ser Ala Gly Glu Gly Ile Thr Ala Tyr Val Val Asp Thr Gly
165 170 175 Ile Arg Val Thr
His Glu Glu Phe Glu Gly Arg Ala Thr Phe Ala Ala 180
185 190 Asn Phe Ile Asp Asp Val Asp Thr Asp
Glu Gln Gly His Gly Ser His 195 200
205 Val Ala Gly Thr Ile Gly Gly Lys Thr Phe Gly Val Ala Lys
Lys Val 210 215 220
Asn Leu Val Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Ser Asn 225
230 235 240 Ser Gly Val Ile Ala
Gly Met Gln Phe Val Ala Ser Asn Ala Thr Ala 245
250 255 Met Gly Leu Lys Gly Arg Ala Val Met Asn
Met Ser Leu Gly Gly Pro 260 265
270 Ala Ser Arg Ala Val Asn Ser Ala Ile Asn Gln Val Glu Ala Ala
Gly 275 280 285 Val
Val Pro Val Val Ala Ala Gly Asn Glu Ser Gln Asp Thr Ala Asn 290
295 300 Thr Ser Pro Gly Ser Ala
Glu Ala Ala Ile Thr Val Gly Ala Ile Asp 305 310
315 320 Gln Thr Asn Asp Arg Met Ala Ser Phe Ser Asn
Phe Gly Glu Leu Val 325 330
335 Asp Ile Phe Ala Pro Gly Val Asn Val Gln Ser Val Gly Ile Arg Ser
340 345 350 Asp Thr
Ser Thr Asn Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His 355
360 365 Val Ala Gly Leu Ala Ala Tyr
Ile Met Ser Leu Glu Asn Ile Thr Gly 370 375
380 Val Gln Ala Val Ser Asp Arg Leu Lys Glu Leu Ala
Gln Ala Thr Gly 385 390 395
400 Ala Arg Ala Arg Gly Val Pro Arg Gly Thr Thr Thr Leu Ile Ala Asn
405 410 415 Asn Gly Phe
Ala 420 159421PRTNeurospora crassa 159Met Val Gly Leu Lys Asn
Val Ala Leu Phe Ala Ala Ser Ile Ile Leu 1 5
10 15 Pro Ala Ser Ile Thr Trp Ala Ala Pro Ile Ile
Glu Val Glu Thr Lys 20 25
30 Pro Ile Pro Glu Lys Tyr Ile Val Leu Leu Lys Pro His Ala Asp
Leu 35 40 45 Glu
Gly His Leu Ser Trp Ala Lys Asp Val His Ala Arg Ser Leu Ser 50
55 60 Arg Arg Asp Thr Ala Gly
Val His Lys Ala Trp Ser Val Gly Ser Lys 65 70
75 80 Phe Lys Ala Tyr Ala Gly Glu Phe Asp Glu Glu
Thr Leu Lys Ile Ile 85 90
95 Gln Arg Asp Glu Arg Asn Val His Ser Ile Glu Pro Asp Lys Ser Trp
100 105 110 Arg Leu
Tyr Lys Ser Asn Lys Lys Asp Asn Asp Asp Ser Asn Ser Asp 115
120 125 Asn Thr Thr Ile Ile Thr Gln
Lys Gln Ala Pro Trp Gly Leu Gly Tyr 130 135
140 Leu Ser His Lys Gly Lys Thr Ser Ser Asp Tyr Val
Tyr Asn Ser Thr 145 150 155
160 Ala Gly Thr Gly Thr Tyr Ala Tyr Val Val Asp Thr Gly Cys Trp Lys
165 170 175 Asp His Val
Glu Phe Glu Gly Arg Val Gln Leu Gly Tyr Asn Ala Tyr 180
185 190 Pro Asp Ser Pro Phe Ile Asp Met
Asp Gly His Gly Thr His Val Thr 195 200
205 Gly Thr Leu Ile Ser Lys Thr Tyr Gly Val Ala Lys Asn
Ala Thr Val 210 215 220
Ile Cys Val Lys Val Phe His Gly Gly Gly Ser Ala Asn Thr Ile Val 225
230 235 240 Met Asp Gly Phe
Glu Trp Ala Val Lys Asp Ile Ile Ala Lys Lys Arg 245
250 255 Gln Arg Asn Ser Val Ile Asn Met Ser
Leu Gly Cys Asp Arg Ser Glu 260 265
270 Ala Phe Asn Ala Ile Val Asp Ala Ala Tyr Asp Gln Gly Ile
Leu Thr 275 280 285
Val Val Ala Ala Gly Asn Glu Asn Gln Pro Ala Ala Leu Val Ser Pro 290
295 300 Ala Ser Ser Ala Arg
Ala Phe Ser Val Gly Ala Ile Asp Asn Lys Asn 305 310
315 320 Thr Arg Ala Tyr Phe Ser Asn Tyr Gly Ala
Ile Val Asp Ile Phe Ala 325 330
335 Pro Gly Val Asn Ile Val Ser Thr Tyr Ile Gly Lys Lys Asp Gly
Asp 340 345 350 Asn
Asn Arg Thr Met Thr Met Ser Gly Thr Ser Met Ala Ser Pro His 355
360 365 Val Ala Gly Leu Ala Leu
Tyr Leu Lys Ser Leu Asp Pro Glu Lys Tyr 370 375
380 Gly Asn Ser Ser Asp Ala His Ser Gly Leu Arg
Ala Leu Gly Val Pro 385 390 395
400 Asp Lys Val Trp Asp Ala Gly Glu Met Ser Pro Asn Leu Val Ala Tyr
405 410 415 Asn Gly
Val Gln Gly 420 160243PRTMyceliophthora thermophila
160Met Lys Leu Ala Val Leu Ile Ala Thr Thr Ala Gly Leu Ala Ala Ala 1
5 10 15 Leu Pro Gln Gly
Val Ala Arg Arg Gly Val Gly Arg Pro Leu His His 20
25 30 Ser Gly Pro Asn Ile Arg Asn Thr Thr
Tyr Pro Gln Tyr Ser Ser Asn 35 40
45 Trp Ala Gly Ala Val Gln Ile Gly Thr Gly Phe Thr Ser Val
Tyr Gly 50 55 60
Thr Ile Thr Val Pro Ser Val His Asp Arg Asn Pro Asn Ala Ala Ala 65
70 75 80 Ser Ala Trp Val Gly
Ile Asp Gly Asp Thr Cys Gln Gln Ala Ile Leu 85
90 95 Gln Thr Gly Val Ser Phe Tyr Gly Asp Gly
Ser Phe Asp Ala Trp Tyr 100 105
110 Glu Trp Ile Pro Asp Tyr Ala Tyr Ser Phe Ser Asn Phe Arg Leu
Ser 115 120 125 Ala
Gly Asp Gln Ile Arg Met Ser Val Glu Ala Ser Ser Lys Arg Ala 130
135 140 Gly Val Ala Thr Leu Glu
Asn Leu Ser Thr Gly Gln Lys Val Ser His 145 150
155 160 Thr Phe Thr Ser Thr Pro Ser Thr Leu Cys Glu
Thr Asn Ala Glu Trp 165 170
175 Ile Val Glu Asp Phe Gln Glu Gly Ser Ser Leu Val Pro Phe Ala Asp
180 185 190 Phe Gly
Thr Val Thr Phe Thr Asp Ala Tyr Ala Thr Gly Ser Ser Gly 195
200 205 Thr Val Thr Pro Ser Gly Ala
Thr Ile Ile Asp Ile Lys Gln Gly Asn 210 215
220 Glu Val Leu Thr Asn Cys Ala Thr Ser Gly Ser Asp
Leu Thr Cys Ser 225 230 235
240 Tyr Thr Gly 161288PRTNeurospora crassa 161Met Lys Leu Leu Ser Pro
Ala Ile Ser Leu Leu Gly Val Ile Ser Gln 1 5
10 15 Pro Ile Leu Ala Gln Phe Thr Phe Thr Ser Thr
Val Glu His Asn Gly 20 25
30 Val Pro Val Pro Gln Ala Glu Thr Asp Leu Lys Pro Phe Lys Pro
Gly 35 40 45 Thr
Leu Gly Arg Ile Arg Ser Arg Thr Asp Asp Asp Ser Gly Pro Glu 50
55 60 Ile Gly Thr Thr Thr Leu
Arg Arg Val Lys Arg Thr Asn Pro Thr Ala 65 70
75 80 Asn Ser Asn Asn Trp Cys Gly Ser Val Gln Ser
Thr Thr Ser Ser Asn 85 90
95 Gln Ile Lys Leu Val His Gly Thr Phe Gln His Pro Thr Cys Thr Gln
100 105 110 Arg Pro
Gly Val Thr Gln Tyr Pro Gln Ala Ala Ala Ala Trp Ile Gly 115
120 125 Ile Asp Gly Asp Ser Trp Thr
Ser Ala Leu Leu Gln Ala Gly Thr Val 130 135
140 Cys Lys Ile Asn Asn Ser Thr Gly Ile Val Glu Asn
Glu Val Trp Trp 145 150 155
160 Gln Trp Val Pro Asn Gly Ala Tyr Thr Ile Thr Asn Ile Pro Val Phe
165 170 175 Ala Gly Asp
Trp Phe Asp Ile Thr Ile Asn Thr Thr Ser Ser Thr Ala 180
185 190 Ala Thr Ile Lys Ile Met Ser Asn
Arg Gly Tyr Thr Tyr Ser Val Asn 195 200
205 Ala Trp Gln Gly Ala Thr Leu Ala Arg Val Asp Ala Asp
Trp Val Val 210 215 220
Glu Arg Pro Tyr Tyr Gly Ser Thr Leu Ala Gly Phe Ala Gln Phe Thr 225
230 235 240 Gln Val Trp Phe
Gln Asn Ala Tyr Ala Thr Leu Thr Ser Gly Thr Ser 245
250 255 Ser Leu Gly Ile Thr Gly Ala Lys Gln
Tyr Gln Ile Pro Gly Gly Cys 260 265
270 Ala Ser Ala Glu Tyr Asp Asn Ser Lys Leu Tyr Ala Ala Val
Ala Ala 275 280 285
162285PRTMyceliophthora thermophila 162Met Trp Ser Ile Val Arg Ser Leu
Ser Leu Ala Ser Leu Ile Ser Ser 1 5 10
15 Ala Cys Thr Val Thr Ala Gln Leu Ser Phe Val Ala Ser
Val Lys Gln 20 25 30
His Gly Lys Asp Val Asp Ala Ser Gly Leu Ser Phe Val Arg Ile Pro
35 40 45 Pro Leu Glu His
Arg Trp His Ala Ser Arg Pro Arg Arg Gly Gln Asn 50
55 60 Asn Arg Thr Val Glu Arg Asp Ala
Val Ser Tyr Ser Ala Asn Trp Cys 65 70
75 80 Gly Ala Ser Gln His Ala Ser Asp Ser Asp Gly Ile
Lys Ser Val Leu 85 90
95 Gly Tyr Phe Thr Ala Pro Asp Leu Thr Leu Arg Pro Gly Thr Pro Ala
100 105 110 Pro Gln Phe
Ala Ala Ala Trp Val Gly Ile Asp Gly Ala Ala Cys Asn 115
120 125 Thr Thr Leu Leu Gln Ala Gly Val
Thr Thr Ile Val Asn Ser Asp Gly 130 135
140 Gly Gln Ser Ala Ser Ala Trp Trp Glu Trp Tyr Pro Glu
Ala Ser Tyr 145 150 155
160 Thr Ile Ser Gly Leu Lys Val Lys Ala Gly Glu Trp Met Ser Val Asn
165 170 175 Ile Thr Thr Lys
Asp Ala Ser Ser Ala Ile Leu Val Ile Glu Asn Ala 180
185 190 Asp Thr Gly Thr Ser Val Thr Leu Glu
Leu Asn Asn Gly Pro Gln Leu 195 200
205 Cys Arg Arg Asp Ala Glu Trp Ile Leu Glu Asp Phe Tyr Glu
Ser Gly 210 215 220
Lys Gln Val Ala Leu Ala Asn Phe Ala Asp Leu Trp Phe Val Asp Ser 225
230 235 240 Gly Ala Thr Thr Val
Gly Gly Lys Asn Val Gly Phe Asp Gly Ala Thr 245
250 255 Met Val His Leu Arg Asp Glu Asn Gly Asn
Val Leu Cys Ser Pro Glu 260 265
270 Pro Tyr Asp Asn Ser Asn Phe Val Val Val Ser Lys Pro
275 280 285 163307PRTMyceliophthora
thermophila 163Met Lys Pro Thr Val Leu Phe Thr Leu Leu Ala Ser Gly Ala
Tyr Ala 1 5 10 15
Ala Ala Thr Pro Ala Ile Pro Gly Tyr Ser Pro Arg Thr Arg Gly Met
20 25 30 Asn Pro His His His
Ala Pro Leu Arg Leu Leu His Thr Phe Thr Pro 35
40 45 Ile Ser Thr Ser Gly Lys Ser Phe Arg
Leu Leu Ala Ser Ser Thr Glu 50 55
60 Ser Thr Lys Gly Gly Ala Ile Leu Gly Leu Pro Asp Asn
Asp Leu Ser 65 70 75
80 Thr Val Arg Thr Thr Ile Arg Ile Pro Ala Ala Lys Met Pro Thr Ala
85 90 95 Gly Pro Thr Ala
Asn Asn Thr Val Gly Glu Tyr Ala Ala Ser Phe Trp 100
105 110 Val Gly Ile Asp Ser Ala Thr Asp Ala
Cys Gly Ala Gly Gly Ser Leu 115 120
125 Arg Ala Gly Val Asp Ile Phe Trp Asp Gly Thr Leu Gly Gly
Gln Gln 130 135 140
Thr Pro Phe Ala Trp Tyr Gln Gly Pro Gly Gln Ala Asp Val Val Gly 145
150 155 160 Phe Gly Gly Gly Phe
Pro Val Gly Glu Gly Asp Leu Val Arg Leu Thr 165
170 175 Leu Glu Ala Gly Pro Ala Gly Gly Glu Glu
Ile Ala Val Val Ala Glu 180 185
190 Asn Phe Gly Arg Asn Val Thr Arg Ala Asp Glu Gly Ala Val Pro
Val 195 200 205 Arg
Lys Val Arg Lys Val Leu Pro Ala Glu Ala Gly Gly Gln Lys Leu 210
215 220 Cys Arg Gly Glu Ala Ala
Trp Met Val Glu Asp Phe Pro Leu Gln Gly 225 230
235 240 Arg Pro Glu Phe Pro Thr Ala Leu Ala Asn Phe
Thr Ser Val Thr Phe 245 250
255 Asn Thr Gly Ile Thr Leu Asp Asp Gly Thr Glu Lys Asp Leu Thr Gly
260 265 270 Ala Glu
Val Leu Asp Ile Gln Leu Glu Ala Gln Gly Gly Arg Leu Thr 275
280 285 Ser Cys Glu Val Val Asp Asp
Arg Asn Val Lys Cys Ala Arg Val Val 290 295
300 Gly Asp Asn 305 164197PRTMyceliophthora
thermophila 164Met Arg Trp Pro Leu Ala Ala Leu Leu Gly Ser Ala Leu Val
Ala Arg 1 5 10 15
Gln Ala Leu Ala Glu Leu Thr Phe Thr Val Glu Ala Thr Arg Asn Gly
20 25 30 Val Pro Ile Pro Ala
Ser Glu Ile Arg Leu Glu Pro Phe Glu Pro Gly 35
40 45 Arg Thr Arg Met Gly Ala Val Ala Glu
Ala Pro Arg Ala Gln Arg Lys 50 55
60 Thr Arg Arg Ser Asn Ala Gln Ala Asp Ser Ala Asn Trp
Cys Gly Ser 65 70 75
80 Val Asn Met Ala Pro Thr Gly Thr Asn Ile Gln Leu Ala His Gly Ser
85 90 95 Phe Gln His Pro
Ser Cys Ser Ile Arg Pro Gly Tyr Thr Phe Pro Gln 100
105 110 Ala Ala Ala Ser Trp Val Gly Ile Asp
Gly Asp Ser Tyr Arg Asp Ala 115 120
125 Leu Leu Gln Ala Gly Thr Val Cys Lys Ile Asp Asn Ser Thr
Gly Val 130 135 140
Val Arg His Glu Ala Trp Trp Gln Trp Val Pro Ser Ala Ala Phe Thr 145
150 155 160 Ile Thr Ser Met Pro
Gly Gln Ser Asn Thr Thr Gly Phe Cys Ile Pro 165
170 175 Tyr Ser Ala Pro Phe Val Ser Leu Cys Phe
Phe Gly Arg Thr Arg Thr 180 185
190 Cys Leu Phe Leu His 195
165621PRTMyceliophthora thermophila 165Met Leu Arg Asn Ile Phe Leu Thr
Ala Ala Leu Ala Ala Phe Gly Gln 1 5 10
15 Cys Gly Ser Thr Val Phe Glu Ser Val Pro Ala Lys Pro
Arg Gly Trp 20 25 30
Thr Arg Leu Gly Asp Ala Ser Ala Asp Gln Pro Leu Arg Leu Arg Ile
35 40 45 Ala Leu Gln Gln
Pro Asn Glu Asp Leu Phe Glu Arg Thr Leu Tyr Glu 50
55 60 Val Ser Asp Pro Ser His Ala Arg
Tyr Gly Gln His Leu Ser Arg Asp 65 70
75 80 Glu Leu Ser Ala Leu Leu Ala Pro Arg Ala Glu Ser
Thr Ala Ala Val 85 90
95 Leu Asn Trp Leu Arg Asp Ala Gly Ile Pro Ser Asp Lys Ile Glu Glu
100 105 110 Asp Gly Glu
Trp Ile Asn Leu Arg Val Thr Val Arg Glu Ala Ser Glu 115
120 125 Leu Leu Asp Ala Asp Phe Gly Val
Trp Ala Tyr Glu Gly Thr Asn Val 130 135
140 Lys Arg Val Arg Ala Leu Gln Tyr Ser Val Pro Glu Glu
Ile Ala Pro 145 150 155
160 His Ile Arg Met Val Ala Pro Val Val Arg Phe Gly Gln Ile Arg Pro
165 170 175 Glu Arg Ser Gln
Val Phe Glu Val Val Glu Thr Ala Pro Ser Gln Val 180
185 190 Lys Val Ala Ala Ala Ile Pro Pro Gln
Asp Leu Asp Val Lys Ala Cys 195 200
205 Asn Thr Ser Ile Thr Pro Glu Cys Leu Arg Ala Leu Tyr Lys
Val Gly 210 215 220
Ser Tyr Gln Ala Glu Pro Ser Lys Lys Ser Leu Phe Gly Val Ala Gly 225
230 235 240 Tyr Leu Glu Gln Trp
Ala Lys Tyr Asp Gln Leu Glu Leu Phe Ala Ser 245
250 255 Thr Tyr Ala Pro Tyr Ala Ala Asp Ala Asn
Phe Thr Ser Val Gly Val 260 265
270 Asn Gly Gly Glu Asn Asn Gln Gly Pro Ser Asp Gln Gly Asp Ile
Glu 275 280 285 Ala
Asn Leu Asp Ile Gln Tyr Ala Val Ala Leu Ser Tyr Lys Thr Pro 290
295 300 Ile Thr Tyr Tyr Ile Thr
Gly Gly Arg Gly Pro Leu Val Pro Asp Leu 305 310
315 320 Asp Gln Pro Asp Pro Asn Asp Val Ser Asn Glu
Pro Tyr Leu Glu Phe 325 330
335 Phe Ser Tyr Leu Leu Lys Leu Pro Asp Ser Glu Leu Pro Gln Thr Leu
340 345 350 Thr Thr
Ser Tyr Gly Glu Asp Glu Gln Ser Val Pro Arg Pro Tyr Ala 355
360 365 Glu Lys Val Cys Gln Met Ile
Gly Gln Leu Gly Ala Arg Gly Val Ser 370 375
380 Val Ile Phe Ser Ser Gly Asp Thr Gly Val Gly Ser
Ala Cys Gln Thr 385 390 395
400 Asn Asp Gly Lys Asn Thr Thr Arg Phe Leu Pro Ile Phe Pro Gly Ala
405 410 415 Cys Pro Tyr
Val Thr Ser Ile Gly Ala Thr Arg Tyr Val Glu Pro Glu 420
425 430 Gln Ala Ala Ala Phe Ser Ser Gly
Gly Phe Ser Asp Ile Phe Lys Arg 435 440
445 Pro Ala Tyr Gln Glu Ala Ala Val Ser Thr Tyr Leu His
Lys His Leu 450 455 460
Gly Ser Arg Trp Lys Gly Leu Tyr Asn Pro Gln Gly Arg Gly Phe Pro 465
470 475 480 Asp Val Ser Ala
Gln Gly Val Ala Tyr His Val Phe Ser Gln Asp Lys 485
490 495 Asp Ile Lys Val Ser Gly Thr Ser Ala
Ser Ala Pro Leu Phe Ala Ala 500 505
510 Leu Val Ser Leu Leu Asn Asn Ala Arg Leu Ala Gln Gly Arg
Pro Pro 515 520 525
Leu Gly Phe Leu Asn Pro Trp Leu Tyr Ser Glu Lys Val Gln Lys Ala 530
535 540 Gly Ala Leu Thr Asp
Ile Val His Gly Gly Ser Ser Gly Cys Thr Gly 545 550
555 560 Lys Asp Met Tyr Ser Gly Leu Pro Thr Pro
Tyr Val Pro Tyr Ala Ser 565 570
575 Trp Asn Ala Thr Pro Gly Trp Asp Pro Val Thr Gly Leu Gly Thr
Pro 580 585 590 Val
Phe Asp Lys Leu Leu Glu Leu Ser Ser Pro Gly Lys Lys Leu Pro 595
600 605 His Ile Gly Gly Gly His
Gly His Gly Ala Gly Gly His 610 615
620 166587PRTNeurospora crassa 166Met Leu Trp Ser Val Leu Leu Leu Ala
Ala Gly Ala Ser Ala His Val 1 5 10
15 Lys Ser Ser Leu Pro Ser Val Pro Ser Gly Trp Lys Lys Val
Arg Ala 20 25 30
Ala Ser Ala Asp Glu Ser Val Ser Leu Lys Ile Ala Leu Pro Ala His
35 40 45 Gln Pro Asp Ala
Leu Glu Thr Ala Ile Leu Arg Val Ser Asp Pro Asn 50
55 60 His His Glu Tyr Gly Met His Leu
Ser Ser Glu Glu Val Arg Ser Leu 65 70
75 80 Val Ala Pro Ala Asp Glu Thr Thr Asp Ala Val Thr
Ser Trp Leu Asn 85 90
95 Arg Asn Gly Ile Lys Gly Lys Val Asp Asn Asp Trp Val Ser Phe Thr
100 105 110 Thr Ser Val
Ala Lys Ala Asn Asn Leu Leu Asn Thr Thr Phe Asp Trp 115
120 125 Tyr Gln Gln Asp Gly Asp Lys Thr
Gly Pro Lys Leu Arg Thr Leu Gln 130 135
140 Tyr Ser Val Pro Asp Glu Leu Asp Ala His Val Asp Met
Ile Gln Pro 145 150 155
160 Thr Thr Arg Phe Gly Lys Leu Ala Ala Lys Ala Ser Thr Ile Phe Glu
165 170 175 Ile Phe Asp Glu
Pro Glu Pro Lys Asn Ile Ala Asn Val Lys Val Gly 180
185 190 Gly Asp His Pro Thr Cys Thr Gly Cys
Ile Tyr Pro Asp Glu Ile Arg 195 200
205 Ser Leu Tyr Asn Ile Lys Tyr Lys Pro Ser Ala Ser Asp Lys
Asn Thr 210 215 220
Ile Ala Phe Ala Ser Tyr Leu Glu Gln Tyr Ser Asn Tyr Asp Asp Phe 225
230 235 240 Thr Ser Phe Ala Lys
Ala Phe Ile Pro Asp Ala Ala Asp Arg Asn Tyr 245
250 255 Thr Val Lys Leu Val Lys Gly Gly Leu His
Asp Gln Ser Pro Asp Lys 260 265
270 Ile Gly Val Glu Ala Asn Leu Asp Leu Gln Tyr Ile Leu Ala Ile
Ser 275 280 285 Asn
Pro Ile Pro Ile Arg Glu Tyr Ser Ile Gly Gly Arg Gly Pro Leu 290
295 300 Val Pro Thr Ala Asn Gln
Pro Gly Pro Glu Ile Ser Asn Glu Pro Tyr 305 310
315 320 Leu Asp Phe Phe Gln Tyr Leu Leu Ser Leu Lys
Asn Ser Glu Leu Pro 325 330
335 Ala Thr Leu Ser Thr Ser Tyr Gly Glu Glu Glu Gln Ser Val Pro Arg
340 345 350 Glu Tyr
Ala Leu Lys Val Cys Ser Met Ile Gly Gln Leu Gly Ala Arg 355
360 365 Gly Val Ser Val Ile Phe Ser
Ser Gly Asp Ser Gly Pro Gly Asp Ala 370 375
380 Cys Ile Arg Asn Asp Gly Thr Asn Ser Thr Tyr Phe
Glu Pro Thr Phe 385 390 395
400 Pro Gly Ala Cys Pro Trp Val Thr Ser Val Gly Gly Thr Tyr Gln Thr
405 410 415 Gly Pro Glu
Lys Ala Val Asp Phe Ser Ser Gly Gly Phe Ser Met Tyr 420
425 430 His Lys Arg Pro Val Tyr Gln Glu
Arg Val Val Lys Lys Tyr Leu Asp 435 440
445 Lys Ile Gly Asp Thr Tyr Ser Asp Phe Phe Asp Glu Gln
Gly Arg Gly 450 455 460
Phe Pro Asp Val Ser Ala Gln Ala Ser Arg Tyr Ala Val Tyr Val Asp 465
470 475 480 Gly Arg Leu Val
Gly Val Ser Gly Thr Ser Ala Ser Ala Pro Met Phe 485
490 495 Ala Gly Leu Val Ala Leu Leu Asn Ala
Ala Arg Lys Ser His Gly Leu 500 505
510 Pro Ser Leu Gly Phe Ile Asn Pro Leu Leu Tyr Ala Ser Lys
Asp Ala 515 520 525
Phe Thr Asp Ile Val Asn Gly Ala Gly Thr Gly Cys Arg Gly Arg Pro 530
535 540 Glu Phe Ala Gly Asp
Val Gly Gly Thr Ala Lys Trp Asn Ala Thr Glu 545 550
555 560 Gly Trp Asp Pro Val Thr Gly Leu Gly Thr
Pro Lys Phe Asp Lys Leu 565 570
575 Leu Ala Leu Ala Ala Pro Gly Val Lys Asn Ala 580
585 167720PRTMyceliophthora thermophila 167Met
Arg Ile Arg His Ala Leu Val Gly Ile Ala Ser Leu Cys Cys Leu 1
5 10 15 Leu Gly Thr Ala Ser Gly
Ala Arg Ile Ser Ser Arg Asp Met Leu Ser 20
25 30 Arg Arg Val Val Pro Pro Ser His Thr Leu
His Glu Arg His Glu Ala 35 40
45 Gly Asn Val Glu Gly Trp Val Lys Arg Gly Leu Ala Asp Ala
Glu Ser 50 55 60
Thr Val Pro Val Arg Ile Gly Leu Lys Gln Ser Asn Val Asp Ala Ala 65
70 75 80 His Asp Leu Leu Met
Asp Ile Ser Asp Pro Arg Ser Pro Asn Tyr Gly 85
90 95 Lys His Leu Ser Arg Ser Glu Val Glu Asp
Leu Phe Ala Pro Arg Glu 100 105
110 His Ser Val Ala Lys Val Lys Arg Trp Leu Ala Ser Ala Gly Val
Asp 115 120 125 Glu
Gly Arg Ile Ser Gln Ser Ala Asn Lys Gln Trp Ile Gln Phe Asp 130
135 140 Ala Pro Val Tyr Glu Leu
Glu Lys Leu Leu Leu Thr Arg Tyr His Ile 145 150
155 160 Phe Glu Asn Leu Glu Thr Gly Val Gln Asn Ile
Ala Cys Ser Glu Tyr 165 170
175 His Val Pro Arg Asp Val Ser His His Ile Asp Tyr Ile Thr Pro Gly
180 185 190 Ile Lys
Leu Met Ala Gly Gly Arg Glu Glu Arg Met Val Arg Trp Arg 195
200 205 Lys Ala Asp Arg Arg Ser Leu
Val Ala Gly Leu Ala Ser Gln Gly Arg 210 215
220 Lys Gly Ala His Gly Met Gly His Gly Gly Gly Gly
Gly Ser Arg Ser 225 230 235
240 Pro Asp Asp Pro Val Val Asp Asp Ser Pro Phe Arg Val Thr Gly Pro
245 250 255 Cys Ser Ala
Glu Ile Thr Pro Asn Cys Ile Arg Ala Gln Tyr Gln Leu 260
265 270 Pro Asn Gly Thr Arg Ala Ala Ser
Gly Asn Glu Leu Gly Ile Phe Gln 275 280
285 Gly Leu Gly Gln His Tyr Ser Gln Glu Asp Leu Asp Asn
Tyr Trp Lys 290 295 300
Tyr Val Ala Pro Trp Val Pro Arg Gly Thr His Pro Glu Leu Arg Ser 305
310 315 320 Ile Asn Gly Ala
Leu Gly Pro Ala Asn Asp Thr Leu Arg Ala Gly Glu 325
330 335 Glu Ala Asp Leu Asp Phe Gln Ile Ala
Ile Pro Leu Ile Trp Pro Gln 340 345
350 Arg Thr Val Leu Phe Gln Thr Asp Asp Glu Trp Tyr Gln Gln
Asp Gln 355 360 365
Gln Arg Ala Asp Thr Lys Tyr Pro Gly Phe Phe Asn Thr Phe Phe Asp 370
375 380 Ala Ile Asp Gly Ser
Tyr Cys His Met Thr Ala Phe Asn Met Thr Gly 385 390
395 400 Asn Cys Val Thr Pro Glu Cys Arg Asp Pro
Glu Tyr Pro Asn Pro Asn 405 410
415 Ala Thr Pro Glu Gln Gly Gly Tyr Ala Gly Ala Leu Met Cys Gly
Arg 420 425 430 His
Arg Pro Thr Ser Val Val Ser Val Ser Tyr Ser Gly Thr Glu Asp 435
440 445 Ser Trp Pro Ala Ser Tyr
Met Arg Arg Gln Cys Leu Glu Val Leu Lys 450 455
460 Leu Ala Leu Gln Gly Val Thr Val Val Glu Ser
Ser Gly Asp Phe Gly 465 470 475
480 Val Gly Gly Arg Pro Phe Asp Pro Arg Ala Gly Cys Leu Gly Pro Asp
485 490 495 Arg Ala
Val Phe Ser Pro Arg Val Met Ala Asn Cys Pro Tyr Val Leu 500
505 510 Ser Val Gly Ala Thr Ala Leu
Val Asp Pro Glu Gln Glu Gln Gln Gln 515 520
525 Gln His Ala Asp Arg Gly Gly Ser Gly Lys Glu Pro
Arg Leu Val Glu 530 535 540
Val Ala Ala Arg Thr Phe Ala Ser Gly Gly Gly Phe Ser Asn Ile Phe 545
550 555 560 Gly Arg Pro
Lys Trp Gln Asp Arg His Val Arg Glu Tyr Leu Arg Lys 565
570 575 Thr Asn Leu Ser Glu Leu Gly Tyr
Asp Asn Ala Ala Gly Met Ser Phe 580 585
590 Asp Ser Leu Arg Pro Pro Pro Ala Gly Gly Lys Leu Phe
Asn Arg Leu 595 600 605
Gly Arg Gly Tyr Pro Asp Val Ala Ala Val Gly Gln Asn Phe Arg Val 610
615 620 Val Leu Arg Gly
Tyr Pro Asn Arg Met His Gly Thr Ser Ala Ala Ala 625 630
635 640 Pro Val Trp Ala Ser Ile Leu Thr Leu
Ile Asn Glu Glu Arg Arg Ala 645 650
655 Val Gly Lys Gly Pro Val Gly Phe Val His Gln Val Leu Tyr
Gln His 660 665 670
Pro Glu Val Phe Thr Asp Ile Thr Val Gly Ser Asn Pro Gly Cys Gly
675 680 685 Thr Asp Gly Phe
Pro Val Glu Glu Gly Trp Asp Pro Val Thr Gly Leu 690
695 700 Gly Ser Pro Ile Tyr Pro Lys Leu
Leu Lys Leu Phe Met Ser Leu Pro 705 710
715 720 168719PRTNeurospora crassa 168Met Phe Arg Phe His
Leu Trp Thr Leu Leu Arg Leu Phe Ala Leu Leu 1 5
10 15 Ser Ser Leu Val Thr Ala Ser Arg Ile Val
Leu Glu Glu Ala Gly His 20 25
30 Leu Pro Ala Gly Trp Lys Val Glu Arg His Ala Thr Ala Ser Asp
Arg 35 40 45 Ile
Gln Leu Ser Ile Ala Leu Lys Glu Pro Gly Ile Glu Glu Leu Lys 50
55 60 Arg Arg Leu Leu Gln Gln
Ser Thr Ser Asp Asp His Pro Asn Ser Arg 65 70
75 80 Gln Phe Thr Lys Glu Glu Val Glu Lys His Arg
Gln Pro Asp Gln Arg 85 90
95 Ser Val Thr Ala Val Gly Arg Trp Leu Gln Ser His Gly Ile Lys Ser
100 105 110 Tyr Asn
Ala Asp Asn Ser Trp Ile Thr Phe Lys Ala Thr Ala Ala Thr 115
120 125 Val Gln Met Leu Phe Glu Ala
Asp Leu Ala Tyr Tyr Ser Tyr Asn Gly 130 135
140 Asp Pro Ser Thr Gln Ile Leu Arg Ser Arg Ser Tyr
Thr Ile Pro Arg 145 150 155
160 Trp Leu Ser Asp Asp Ile Asp Phe Val His Pro Leu Thr Asn Phe Met
165 170 175 Pro Pro Arg
Asn Arg Asn Asp Gly Thr Leu Gly Ile Gly Arg Arg Gln 180
185 190 Pro Ile Gln Pro Lys Leu Ser Ala
Arg Glu Asp Phe Phe Ala Pro Pro 195 200
205 Cys Trp Thr Gly Thr Phe Pro Gly Cys Ile Arg Lys Leu
Tyr Asn Leu 210 215 220
Thr Tyr Thr Pro Ser Pro Asp Phe Arg Ser Pro Ser Pro Val Arg Phe 225
230 235 240 Gly Ile Ala Ser
Phe Leu Glu Gln Tyr Ile Thr His Arg Asp Val Thr 245
250 255 Ser Phe Leu Ala Thr Tyr Ala Arg Glu
Leu Leu Pro Leu Arg Pro Thr 260 265
270 Pro Ser Arg Gly Gly Ser Gly Gly Ser Leu Thr Leu Pro Pro
Val Thr 275 280 285
Asn Thr Thr Ser Glu Pro Pro Tyr Asn Ile Thr Ile Thr Leu Leu Asn 290
295 300 Asn Ala Thr Arg Trp
Asp Pro His Ser Thr Asp Pro Ala Leu Ser Gly 305 310
315 320 Leu Glu Ala Asn Leu Asp Val Gln Tyr Ala
Leu Ser Leu Gly His Pro 325 330
335 Thr Arg Val Ile Tyr Tyr Ala Thr Gly Gly Arg Gly Thr Lys Leu
Asp 340 345 350 Ser
Ser Gly Arg Pro Leu Pro Thr Asn Asp Pro Arg Ala Asn Asn Glu 355
360 365 Pro Phe Leu Glu Phe Leu
Gln Ala Leu Leu Ala Leu Pro Asp Asn Gln 370 375
380 Ile Pro His Val Leu Ser Ile Ser Tyr Ala Asp
Asp Glu Gln Ser Val 385 390 395
400 Pro Arg Lys Tyr Ala His Arg Val Cys Asp Leu Phe Ala Ala Val Ala
405 410 415 Ala Arg
Gly Thr Ser Val Leu Val Ala Thr Gly Asp Gly Gly Ala Ala 420
425 430 Gly Ile Gly Phe Ser Ala Gly
Gly Gly Asp Thr Cys Ile Lys Asn Asp 435 440
445 Gly Ser Gly Arg Arg Ala Phe Val Pro Thr Phe Pro
Ala Ser Cys Pro 450 455 460
Trp Val Thr Ser Val Gly Ala Thr Asp Asn Thr Ala Leu Asn Leu Thr 465
470 475 480 Gly Ala Ala
Phe Ser Ser Gly Gly Phe Ser Glu Tyr Phe Asp Arg Pro 485
490 495 Leu Trp Gln Arg Ala Ala Val Asp
Pro Tyr Val Ser Ser Leu Leu Arg 500 505
510 Ser Arg Ser Ser Lys Pro Gly Gln Pro Ser Gln Pro Arg
Asp Leu Lys 515 520 525
Gly Val Tyr Phe Ser His Asn Gly Arg Gly Met Pro Asp Met Ala Ala 530
535 540 Ile Gly Ser Gly
Phe Gln Ile Ile His Arg Gly Glu Met Val Glu Val 545 550
555 560 Arg Gly Thr Ser Ala Ser Thr Pro Val
Val Ala Ala Met Val Ala Leu 565 570
575 Val Asn Asp Gln Arg Leu Arg Gln Gly Lys Arg Ser Leu Gly
Trp Leu 580 585 590
Asn Gly His Leu Tyr Leu Asp Pro Arg Val Arg Arg Val Leu Thr Asp
595 600 605 Val Lys Trp Gly
Arg Ser Glu Gly Cys Val Phe Pro Gly Glu Ala Leu 610
615 620 Glu Glu Gly Arg Gly Lys Gly Lys
Glu Lys Tyr Trp Arg His Ser Val 625 630
635 640 Val Glu Lys Arg Gln Gly Asn Ser Glu Glu Asp Gly
Gly Thr His Gly 645 650
655 Gly Asp Gly Glu Gly Lys Ala Asp Glu Glu Asp Trp Gly Gly Glu Gly
660 665 670 Glu Val Gly
Glu Gly Glu Gly Asp Gln Ser Glu Asn Val Ile Leu Gly 675
680 685 Gly Trp Asp Ala Arg Lys Gly Trp
Asp Pro Val Thr Gly Leu Gly Val 690 695
700 Pro Gly Asp Phe Gln Glu Met Leu Lys Val Leu Gly Ser
Val Trp 705 710 715
169454PRTNeurospora crassa 169Met Arg Ala Thr Leu Val Val Val Leu Cys His
Leu Ser Leu Ala Phe 1 5 10
15 Ala Leu Ala Ile Ser Pro Ala Ala Ser His Trp Lys Arg Ser Ala Arg
20 25 30 Leu Ala
Ser Asp Gln Thr Ala Ser Glu Arg Tyr Ser Leu Pro Ser Arg 35
40 45 Val Ala Arg Tyr Ile Asp Tyr
Val Leu Pro Ala Pro Asp Pro Asp Pro 50 55
60 Val Ser Ser Ala Pro Lys Ser Val Ala Val Gln Asp
Pro Pro Thr Leu 65 70 75
80 Lys Gly Val Ile Gly Ala Arg Gln Thr Arg Asp Val Asp Cys Leu Gln
85 90 95 Tyr Ile Ala
Pro Gln Cys Leu Arg Gln Leu Ala Trp Leu Ala Glu Asp 100
105 110 Leu Asp Met Phe Phe Gly Asp Phe
Ala Pro Asp Leu Leu Thr Asn Phe 115 120
125 Asn Leu Glu Pro Asn Leu Asp Tyr Lys Tyr Thr Met Ala
Met Ala Lys 130 135 140
Pro Ile Pro Val Thr Asn Ile Gln Val Gly Asp Phe Val Val Gln Gly 145
150 155 160 Asn Met Asn Ile
Met Leu Ala Ala Phe Asn Ala His Tyr Cys Arg Thr 165
170 175 Gly Leu Asp Pro Gln Phe Asp Pro Val
Tyr Pro Asn Pro Ala Pro Gly 180 185
190 Gly Tyr Asn Ala Ser Asp Cys Gly Thr His Val Pro Pro Arg
Val Ile 195 200 205
Ala Ile Met Tyr Ala Trp Asn Lys Ala Trp Tyr Ser Asp Ala Asp Phe 210
215 220 Ala Ser Ile Phe Pro
Ala Ser Asp Pro Trp Val Thr Ser Val Gly Gly 225 230
235 240 Thr Gln Phe Leu Pro Val Val Ser Asn Gly
Ser Ser Ser Thr Thr Ala 245 250
255 Ser Ser Gly Met Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser 260 265 270 Ser
Ser Ser Ser Ser Ser Ser Ser Ser Leu Phe Pro Gly Glu Thr Ala 275
280 285 Leu Asp Asp Asn Asn Thr
Gly Ser Ser Gly Gly Ser Phe Ser Arg Leu 290 295
300 Phe Pro Gly Pro Trp Tyr Gln Gly Asn Leu Thr
Arg Glu Tyr Leu Ala 305 310 315
320 Ser Ala Pro Gly Ala Ala Glu Leu Ala Arg Gln Gly Tyr Phe Asn Gly
325 330 335 Ser Gly
Arg Gly Tyr Pro Asp Ile Ser Ala Met Ala Arg Ser Phe Leu 340
345 350 Val Ala Leu His Gly Gly Tyr
His Ala Val Ser Gly Thr Ser Ala Ser 355 360
365 Thr Pro Val Val Ala Ala Met Val Ala Lys Ile Asn
Asp Ala Arg Leu 370 375 380
His Ala Gly Lys Ser Thr Val Gly Phe Leu Asn Pro Val Leu Tyr Ser 385
390 395 400 Ala Ala Ala
Gly Lys Ala Gly Val Leu Arg Asp Val Pro Leu Gly Lys 405
410 415 Asn His Asp Cys Gly Val Gly Glu
Ala Phe Pro Ala Arg Arg Ala Trp 420 425
430 Asp Ala Val Thr Gly Leu Gly Thr Pro Asp Phe Glu Lys
Leu Lys Glu 435 440 445
Leu Tyr Leu Gly Leu Pro 450 17050PRTAspergillus
niger 170Ile Val Thr Trp Asp Glu Ala His Phe Gly Lys Phe Gly Ser His Tyr
1 5 10 15 Leu Lys
Arg Glu Phe Tyr Phe Asp Val His Pro Pro Leu Gly Lys Met 20
25 30 Leu Val Gly Leu Ser Gly Phe
Leu Ala Gly Tyr Asn Gly Ser Phe Glu 35 40
45 Phe Lys 50 17150PRTAspergillus oryzae
171Ile Val Thr Trp Asp Glu Ala His Phe Gly Lys Phe Gly Ser His Tyr 1
5 10 15 Leu Lys Arg Glu
Phe Tyr Phe Asp Val His Pro Pro Leu Gly Lys Met 20
25 30 Leu Val Gly Leu Ser Gly Tyr Leu Ala
Gly Tyr Asn Gly Ser Phe Glu 35 40
45 Phe Lys 50 17250PRTAspergillus nidulans 172Ile Val
Thr Trp Asp Glu Ala His Phe Gly Lys Phe Gly Ser His Tyr 1 5
10 15 Leu Lys Arg Glu Phe Tyr Phe
Asp Val His Pro Pro Leu Gly Lys Met 20 25
30 Leu Val Gly Leu Ser Gly Leu Leu Ala Gly Tyr Asn
Gly Ser Phe Glu 35 40 45
Phe Lys 50 17350PRTMyceliophthora thermophila 173Ile Val Thr
Trp Asp Glu Ala His Phe Gly Lys Phe Gly Ser His Tyr 1 5
10 15 Leu Lys Arg Glu Phe Tyr Phe Asp
Val His Pro Pro Ala Gly Lys Leu 20 25
30 Leu Val Gly Leu Ser Gly Tyr Leu Ala Gly Tyr Asn Gly
Ser Phe Glu 35 40 45
Phe Lys 50 17450PRTNeurospora crassa 174Ile Val Thr Trp Asp Glu
Ala His Phe Gly Lys Phe Gly Ser His Tyr 1 5
10 15 Leu Lys Arg Glu Phe Tyr Phe Asp Val His Pro
Pro Ala Gly Lys Leu 20 25
30 Leu Val Gly Leu Ser Gly Leu Leu Ala Gly Tyr Asn Gly Ser Phe
Glu 35 40 45 Phe
Lys 50 17550PRTTrichoderma virens 175Ile Val Thr Trp Asp Glu Ala His
Phe Gly Lys Phe Gly Ser Tyr Tyr 1 5 10
15 Ile Lys His Glu Tyr Tyr Phe Asp Val His Pro Pro Leu
Gly Lys Met 20 25 30
Leu Val Gly Leu Ser Gly Val Leu Ala Gly Tyr Asn Gly Ser Phe Glu
35 40 45 Phe Lys 50
17650PRTTrichoderma reesei 176Ile Val Thr Trp Asp Glu Ala His Phe Gly Lys
Phe Gly Ser Tyr Tyr 1 5 10
15 Ile Lys His Glu Tyr Tyr Phe Asp Val His Pro Pro Leu Gly Lys Met
20 25 30 Leu Val
Gly Leu Ser Gly Val Leu Ala Gly Tyr Asn Gly Ser Phe Glu 35
40 45 Phe Lys 50
17750PRTFusarium oxysporum 177Ile Val Thr Trp Asp Glu Ala His Phe Gly Lys
Phe Gly Ser Tyr Tyr 1 5 10
15 Ile Lys His Glu Tyr Tyr Phe Asp Val His Pro Pro Leu Gly Lys Met
20 25 30 Leu Val
Gly Leu Ser Gly Val Leu Ala Gly Tyr Asn Gly Thr Phe Glu 35
40 45 Phe Lys 50
17850PRTNeurospora crassa 178Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Lys Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Leu
20 25 30 Met Ile
Thr Leu Phe Gly Trp Leu Ala Gly Phe Asp Gly Ser Phe Asp 35
40 45 Phe Lys 50
17950PRTMyceliophthora thermophila 179Ser Val Val Phe Asp Glu Val His Phe
Gly Gly Phe Ala Thr Lys Tyr 1 5 10
15 Ile Lys Gly Lys Phe Phe Met Asp Val His Pro Pro Leu Ala
Lys Leu 20 25 30
Met Ile Thr Leu Phe Gly Trp Leu Ala Gly Phe Lys Gly Asn Phe Asp
35 40 45 Phe Lys 50
18050PRTTrichoderma virens 180Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Lys Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Met
20 25 30 Leu Ile
Ala Leu Thr Gly Trp Leu Ala Gly Phe Asp Gly Asn Phe Asp 35
40 45 Phe Lys 50
18150PRTTrichoderma atroviride 181Ser Val Val Phe Asp Glu Val His Phe Gly
Gly Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala Lys
Met 20 25 30 Leu
Ile Ala Leu Thr Gly Trp Leu Ala Gly Phe Asp Gly Asp Phe Asp 35
40 45 Phe Lys 50
18250PRTTrichoderma reesei 182Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Met
20 25 30 Leu Ile
Ala Leu Thr Gly Trp Leu Ala Gly Phe Asp Gly Asn Phe Asp 35
40 45 Phe Lys 50
18350PRTFusarium oxysporum 183Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Thr Lys Tyr 1 5 10
15 Ile Lys Gly Lys Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Met
20 25 30 Leu Ile
Ala Leu Thr Gly Trp Leu Ala Gly Phe Asp Gly Ser Phe Asp 35
40 45 Phe Lys 50
18450PRTAspergillus nidulans 184Ser Val Val Phe Asp Glu Val His Phe Gly
Gly Phe Ala Thr Lys Tyr 1 5 10
15 Ile Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala Lys
Leu 20 25 30 Leu
Ile Thr Leu Ala Gly Trp Leu Ala Gly Phe Lys Gly Asp Phe Asp 35
40 45 Phe Lys 50
18550PRTAspergillus oryzae 185Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Leu
20 25 30 Leu Ile
Thr Leu Ala Gly Trp Leu Ala Gly Phe Asn Gly Asp Phe Asp 35
40 45 Phe Lys 50
18650PRTAspergillus niger 186Ser Val Val Phe Asp Glu Val His Phe Gly Gly
Phe Ala Thr Lys Tyr 1 5 10
15 Ile Lys Gly Arg Phe Phe Met Asp Val His Pro Pro Leu Ala Lys Leu
20 25 30 Leu Ile
Thr Leu Ala Gly Trp Leu Ala Gly Phe Asp Gly Glu Phe Asp 35
40 45 Phe Lys 50
18750PRTPenicillium chrysogenum 187Ser Val Val Phe Asp Glu Val His Phe
Gly Gly Phe Ala Ser Lys Tyr 1 5 10
15 Ile Lys Gly Lys Phe Phe Met Asp Val His Pro Pro Leu Ala
Lys Leu 20 25 30
Leu Leu Thr Leu Ala Gly Trp Leu Ala Gly Phe Asp Gly Asn Phe Asp
35 40 45 Phe Lys 50
18850PRTTrichoderma reesei 188Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Ala Lys Leu
20 25 30 Leu Phe
Ala Phe Val Gly Trp Leu Val Gly Tyr Asp Gly His Phe His 35
40 45 Phe Asp 50
18950PRTTrichoderma virens 189Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Ala Lys Leu
20 25 30 Leu Phe
Ala Phe Val Gly Trp Leu Val Gly Tyr Asp Gly His Phe His 35
40 45 Phe Glu 50
19050PRTFusarium oxysporum 190Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Glu Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu
20 25 30 Leu Phe
Ala Phe Val Gly Trp Leu Val Gly Tyr Asp Gly Asn Phe His 35
40 45 Phe Glu 50
19150PRTGibberella zeae 191Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Glu Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu
20 25 30 Leu Phe
Ala Phe Val Gly Trp Leu Val Gly Tyr Asp Gly His Phe His 35
40 45 Phe Asp 50
19250PRTMyceliophthora thermophila 192Glu Val Val Phe Asp Glu Val His Phe
Gly Lys Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Glu Arg Thr Tyr Phe Phe Asp Val His Pro Pro Leu Gly
Lys Leu 20 25 30
Leu Phe Ala Phe Met Gly Trp Leu Val Gly Tyr Asp Gly His Phe His
35 40 45 Phe Glu 50
19350PRTNeurospora crassa 193Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Glu Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu
20 25 30 Leu Phe
Ala Phe Met Gly Trp Leu Val Gly Tyr Asp Gly His Phe His 35
40 45 Phe Glu 50
19450PRTAspergillus nidulans 194Gln Val Val Phe Asp Glu Val His Phe Gly
Lys Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Arg Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Ala Lys
Leu 20 25 30 Leu
Leu Ala Phe Thr Gly Trp Leu Val Gly Tyr Asp Gly His Phe Leu 35
40 45 Phe Glu 50
19550PRTAspergillus niger 195Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu
20 25 30 Leu Phe
Ala Phe Met Gly Trp Leu Val Gly Tyr Asp Gly His Phe Leu 35
40 45 Phe Asp 50
19650PRTAspergillus oryzae 196Glu Val Val Phe Asp Glu Val His Phe Gly Lys
Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly Lys Leu
20 25 30 Leu Phe
Ala Ala Val Gly Trp Leu Ile Gly Tyr Asp Gly His Phe Leu 35
40 45 Phe Glu 50
19750PRTPenicillium chrysogenum 197Glu Val Val Phe Asp Glu Val His Phe
Gly Lys Phe Ala Ser Tyr Tyr 1 5 10
15 Leu Gln Arg Thr Tyr Phe Phe Asp Val His Pro Pro Phe Gly
Lys Leu 20 25 30
Leu Phe Ala Leu Met Gly Trp Leu Val Gly Phe Asp Gly Ser Phe Leu
35 40 45 Phe Glu 50
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