TITLE OF THE INVENTION

NOVEL CELL WALL DECONSTRUCTION ENZYMES OF AMORPHOTHECA RESINAE, RHIZOMUCOR PUSILLUS, AND CALCARISPORIELLA THERMOPHILA, AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to novel polypeptides and enzymes having activities relating to biomass processing and/or degradation (e.g., cell wall deconstruction), as well as polynucleotides, vectors, cells, compositions and tools relating to same, or functional variants thereof. More particularly, the present invention relates to secreted enzymes that may be isolated from the fungi Amorphotheca resinae strain DAOM 194228, Rhizomucor pusillus strain CBS 183.67, and Calcarisporiella thermophila strain CBS 279.70. Uses thereof in various industrial processes such as in biofuels, food preparation, animal feed, pulp and paper, textiles, detergents, waste treatment and others are also disclosed.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form entitled "Seq_Listing_AMORE_RHIPU_CALTH.txt ", created January 10, 2014 having a size of about 4.17 MB. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] Biomass-processing enzymes have a number of industrial applications such as in: the biofuel industry (e.g., improving ethanol yield and/or increasing the efficiency and economy of ethanol production); the food industry (e.g., production of cereal-based food products; the feed-enzyme industry (e.g., increasing the digestibility/absorption of nutrients); the pulp and paper industry (e.g., enhancing bleachability of pulp); the textile industry (e.g., treatment of cellulose-based fabrics); the waste treatment industry (e.g., de-colorization of synthetic dyes); the detergent industry (e.g., providing eco-friendly cleaning products); and the rubber industry (e.g., catalyzing the conversion of latex into foam rubber).

[0004] In particular, driven by the limited availability of fossil fuels, there is a growing interest in the biofuel industry for improving the conversion of biomass into second-generation biofuels. This process is heavily dependent on inexpensive and effective enzymes for the conversion of lignocellulose to ethanol. Cellulase enzyme cocktails involve the concerted action of endoglucanases, cellobiohydrolases (also known as exoglucanases), and beta-glucosidases. The current cost of cellulose-degrading enzymes is too high for bioethanol to compete economically with fossil fuels. Cost reduction may result from the discovery of cellulase enzymes with, for example, higher specific activity, lower production costs, and/or greater compatibility with processing conditions including temperature, pH and the presence of inhibitors in the biomass, or produced as the result of biomass pre-treatment.

[0005] Conversion of plant biomass to glucose may also be enhanced by supplementing cellulose cocktails with enzymes that degrade the other components of biomass, including hemicelluloses, pectins and lignins, and their linkages, thereby improving the accessibility of cellulose to the cellulase enzymes. Such enzymes include, without being limiting, to: xylanases, mannanases, arabinanases, esterases, glucuronidases, xyloglucanases and arabinofuranosidases for hemicelluloses; lignin peroxidases, manganese-dependent peroxidases, versatile peroxidases, and laccases for lignin; and pectate lyase, pectin lyase, polygalacturonase, pectin acetyl esterase, alpha-arabinofuranosidase, beta-galactosidase, galactanase, arabinanase, rhamnogalacturonase, rhamnogalacturonan lyase, and rhamnogalacturonan acetyl esterase, xylogalacturonosidase, xylogalacturonase, and rhamnogalacturonan lyase. Additionally, glycoside hydrolase family 61 (GH61) proteins have been shown to stimulate the activity of cellulase preparations.

[0006] These enzymes may also be useful for other purposes in processing biomass. For example, the lignin modifiying enzymes may be used to alter the structure of lignin to produce novel materials, and hemicellulases may be employed to produce 5-carbon sugars from hemicelluloses, which may then be further converted to chemical products.

[0007] There is also a growing need for improved enzymes for food processing and feed applications. Cereal-based food products such as pasta, noodles and bread can be prepared from dough which is usually made from the basic ingredients (cereal) flour, water and optionally salt. As a result of a consumer-driven need to replace the chemical additives by more natural products, several enzymes have been developed with dough and/or cereal-based food product-improving properties, which are used in all possible combinations depending on the specific application conditions. Suitable enzymes include, for example, xylanase, starch degrading enzymes, oxidizing enzymes, fatty material splitting enzymes, protein degrading, and modifying or crosslinking enzymes. Many of these enzymes are also used for treating animal feed or animal feed additives, to make them more digestible or to improve their nutritional quality. Amylases are used for the conversion of plant starches to glucose. Pectin-active enzymes are used in fruit processing, for example to increase the yield of juices, and in fruit juice clarification, as well as in other food processing steps.

[0008] There is also a growing need for improved enzymes in other industries. In the pulp and paper industry, enzymes are used to make the bleaching process more effective and to reduce the use of oxidative chemicals. In the textile industry, enzymatic treatment is often used in place of (or in addition to) a bleaching treatment to achieve a "used" look of jeans, and can also improve the softness/feel of fabrics. When used in detergent compositions, enzymes can enhance cleaning ability or act as a softening agent. In the waste treatment industry, enzymes play an important role in changing the characteristics of the waste, for example, to become more amenable to further treatment and/or for bio-conversion to value-added products.

[0009] There is also a growing need for indutrial enzymes and proteins that are "thermostable" in that they retain a level of their function or protein activity at temperatures about 50°C. These thermostable enzymes are highly desirable, for example, to be able to perform reactions at elevated temperatures to avoid or reduce contamination by microorganisms (e.g., bacteria).

[0010] There thus remains a need in the above-mentioned industries and others for biomass-processing enzymes, polynucleotides encoding same, and recombinant vectors and strains for expressing same. [0011] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0012] In general, the present invention relates to soluble, secreted proteins relating to biomass processing and/or degradation (e.g., cell wall deconstruction) that may be isolated from the fungi Amorphotheca resinae strain DAOM 194228, Rhizomucor pusillus strain CBS 183.67, and Calcarisporiella thermophila strain CBS 279.70, as well as polynucleotides, vectors, compositions, cells, antibodies, kits, products and uses associated with same. Briefly, these fungal strains were cultured in vitro and genomic DNA along with total RNA were isolated therefrom. These nucleic acids were then used to determine/assemble fungal genomic sequences and generate cDNA libraries. Bioinformatic tools were used to predict genes in the assembled genomic sequences, and those genes encoding proteins relating to biomass-degradation (e.g., cell wall deconstruction) were identified based on bioinformatics (e.g., the presence of conserved domains). Sequences predicted to encode proteins which are targeted to the mitochondria or bound to the cell wall were removed. cDNA clones comprising full-length sequences predicted to encode soluble, secreted proteins relating to biomass-degradation were fully sequenced and cloned into appropriate expression vectors for protein production and characterization. The full-length genomic, exonic, intronic, coding and polypeptide sequences are disclosed herein, along with corresponding putative (biological) functions and/or protein activities, where available.

[0013] The soluble, secreted, biomass degradation proteins of the present invention comprise a proteome which is referred to herein as the SSBD proteome of Amorphotheca resinae, Rhizomucor pusillus, or Calcarisporiella thermophila.

[0014] Accordingly, in some aspects the present invention relates to an isolated polypeptide which is:

(a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 389-582, 883- 1032, and 1373-1542;

(b) a polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the polypeptide defined in (a);

(c) a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence of any one of SEQ ID NOs: 195-388, 733-882, and 1203-1372;

(d) a polypeptide comprising an amino acid sequence encoded by any one the exonic nucleic acid sequences corresponding to the positions as defined in Tables 2A-2C;

(e) a polypeptide comprising an amino acid sequence encoded by a polynucleotide molecule that hybridizes under medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of a polynucleotide molecule comprising the nucleic acid sequence defined in (c) or (d); (f) a polypeptide comprising an amino acid sequence encoded by a polynucleotide molecule having at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity to a polynucleotide molecule comprising the nucleic acid sequence defined in (c) or (d);

(g) a functional variant of the polypeptide defined in (a) comprising a substitution, deletion, and/or insertion at one or more residues; or

(h) a functional fragment of the polypeptide of any one of (a) to (g).

[0015] In some embodiments, the above mentioned polypeptide has a corresponding function and/or protein activity according to Tables 1A-1 C.

[0016] In some embodiments, the above mentioned polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 389-582, 883-1032, and 1373-1542.

[0017] In some embodiments, the above mentioned polypeptide is a recombinant polypeptide.

[0018] In some embodiments, the above mentioned polypeptide is obtainable from a fungus. In some embodiments, the fungus is from the genus Amorphotheca, Rhizomucor, or Calcarisporiella. In some embodiments, the fungus is Amorphotheca resinae, Rhizomucor pusillus, or Calcarisporiella thermophila.

[0019] In some aspects, the present invention relates to an antibody that specifically binds to any one of the above mentioned polypeptides.

[0020] In some aspects, the present invention relates to an isolated polynucleotide molecule encoding any one of the above mentioned polypeptides.

[0021] In some aspects, the present invention relates to an isolated polynucleotide molecule which is:

(a) a polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 389-582, 883-1032, and 1373-1542;

(b) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 1- 194, 583-732, and 1033-1202;

(c) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs:

195-388, 733-882, and 1203-1372;

(d) a polynucleotide molecule comprising any one of the exonic nucleic acid sequences corresponding to the positions as defined in Tables 2A-2C;

(e) a polynucleotide molecule comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity to any one of the polynucleotide molecules defined in (a) to (d); or

(f) a polynucleotide molecule that hybridizes under medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of any one of the polynucleotide molecules defined in (a) to (e). [0022] In some embodiments, the above mentioned polynucleotide molecule is obtainable from a fungus. In some embodiments, the fungus is from the genus Amorphotheca, Rhizomucor, or Calcarisporiella. In some embodiments, the fungus is Amorphotheca resinae, Rhizomucor pusillus, or Calcarisporiella thermophila.

[0023] In some aspects, the present invention relates to a vector comprising any one of the above mentioned polynucleotide molecules. In some embodiments, the vector comprises a regulatory sequence operatively linked to the polynucleotide molecule for expression of same in a suitable host cell. In some embodiments, the suitable host cell is a bacterial cell; a fungal cell; or a filamentous fungal cell.

[0024] In some embodiments, the present invention relates to a recombinant host cell comprising any one of the above mentioned polynucleotide molecules or vectors. In some embodiments, the present invention relates to a polypeptide obtainable by expressing the above mentioned polynucleotide or vector in a suitable host cell. In some embodiments, the suitable host cell is a bacterial cell; a fungal cell; or a filamentous fungal cell.

[0025] In some aspects, the present invention relates to a composition comprising any one of the above mentioned polypeptides or the recombinant host cells. In some embodiments, the composition further comprises a suitable carrier. In some embodiments, the composition further comprises a substrate of the polypeptide. In some embodiments, the substrate is biomass.

[0026] In some aspects, the present invention relates to a method for producing any one of the above mentioned polypeptides, the method comprising: (a) culturing a strain comprising the above mentioned polynucleotide molecule or vector under conditions conducive for the production of the polypeptide; and (b) recovering the polypeptide. In some embodiments, the strain is a bacterial strain; a fungal strain; or a filamentous fungal strain.

[0027] In some aspects, the present invention relates to a method for producing any one of the above mentioned polypeptides, the method comprising: (a) culturing the above mentioned recombinant host cell under conditions conducive for the production of the polypeptide; and (b) recovering the polypeptide.

[0028] In some aspects, the present invention relates to a method for preparing a food product, the method comprising incorporating any one of the above mentioned polypeptides during preparation of the food product. In some embodiments, the food product is a bakery product.

[0029] In some aspects, the present invention relates to the use of the above mentioned polypeptide for the preparation or processing of a food product. In some aspects, the present invention relates to the above mentioned polypeptide for use in the preparation or processing of a food product. In some embodiments, the above mentioned food product is a bakery product.

[0030] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for the preparation of animal feed. In some aspects the present invention relates to the use of any one of the above mentioned polypeptides for increasing digestion or absorption of animal feed. In some aspects, the present invention relates to any one of the above mentioned polypeptides for use in the preparation of animal feed, or for increasing digestion or absorption of animal feed. In some embodiment, the animal feed is a cereal- based feed. [0031] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for the production or processing of kraft pulp or paper. In some aspects, the present invention relates to any one of the above mentioned polypeptides for the production or processing of kraft pulp or paper. In some embodiments, the processing comprises prebleaching and/or de-inking.

[0032] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for processing lignin. In some aspects the present invention relates to any one of the above mentioned polypeptides for processing lignin.

[0033] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for producing ethanol. In some aspects the present invention relates to any one of the above mentioned polypeptides for producing ethanol.

[0034] In some embodiments, the above mentioned uses are in conjunction with cellulose or a cellulase.

[0035] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for treating textiles or dyed textiles. In some aspects, the present invention relates to any one of the above mentioned polypeptides for treating textiles or dyed textiles.

[0036] In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for degrading biomass or pretreated biomass. In some aspects the present invention relates to any one of the above mentioned polypeptides for degrading biomass or pretreated biomass.

[0037] In some embodiments, the present invention relates to proteins and/or enzymes that are thermostable. In some embodiments, a polypeptide of the present invention retains a level of its function and/or protein activity at about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, or about 95°C. In some embodiments, a polypeptide of the present invention retains a level of its function and/or protein activity between about 50°C and about 95°C, between about 50°C and about 90°C, between about 50°C and about 85°C, between about 50°C and about 80°C, between about 50°C and about 75°C, between about 50°C and about 70°C, or between about 50°C and about 65°C. In some embodiments, a polypeptide of the present invention has optimal or maximal function and/or protein activity greater than 50°C, greater than 55°C, greater than 60°C, greater than 65°C, or greater than 70°C. In some embodiments, a polypeptide of the present invention has optimal or maximal function and/or protein activity between about 50°C and about 95°C, between about 50°C and about 90°C, between about 50°C and about 85°C, between about 50°C and about 80°C, between about 50°C and about 75°C, between about 50°C and about 70°C, or between about 50°C and about 65°C.

[0038] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Commonly understood definitions of molecular biology terms can be found for example in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991 , Harper Perennial, New York, NY), Rieger et al., Glossary of genetics: Classical and molecular, 5 ^th edition, Springer-Verlag, New-York, 1991 ; Alberts et al., Molecular Biology of the Cell, 4 ^th edition, Garland science, New-York, 2002; and, Lewin, Genes VII, Oxford University Press, New-York, 2000. Generally, the procedures of molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al., (2000, Molecular Cloning - A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New-York).

[0039] Further objects and advantages of the present invention will be clear from the description that follows.

Definitions

[0040] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0041] In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0042] Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one-letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the lUPAC-IUB Biochemical Nomenclature Commission.

[0043] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0044] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.

[0045] The term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about". Unless indicated otherwise, use of the term "about" before a range applies to both ends of the range.

[0046] The term "DNA" or "RNA" molecule or sequence (as well as sometimes the term "oligonucleotide") refers to a molecule comprised generally of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C). In "RNA", T is replaced by uracil (U).

[0047] The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency. [0048] As used herein, "polynucleotide" or "nucleic acid molecule" refers to a polymer of nucleotides and includes DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA), and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single- stranded (coding strand or non-coding strand [antisense]). Conventional deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are included in the terms "nucleic acid molecule" and "polynucleotide" as are analogs thereof (e.g., generated using nucleotide analogs, e.g., inosine or phosphorothioate nucleotides). Such nucleotide analogs can be used, for example, to prepare polynucleotides that have altered base-pairing abilities or increased resistance to nucleases. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-0- methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2' halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see "The Biochemistry of the Nucleic Acids 5-36", Adams et al., ed., 11 th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481 ). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).

[0049] An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes, but should not limited to DNA and RNA. The "isolated" nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized.

[0050] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, and very often include an open reading frame encoding a protein, e.g., polypeptides of the present invention. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences, as well known.

[0051] "Amplification" refers to any in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. In vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand- displacement amplification (SDA including multiple strand-displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as QB-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0320308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Two other known strand- displacement amplification methods do not require endonuclease nicking (Dattagupta et al., U.S. Patent No. 6,087,133 and U.S. Patent No. 6,124,120 (MSDA)). Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase (e.g., see Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 25 and Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173 1 177; Lizardi et al., 1988, BioTechnology 6:1 197 1202; Malek et al., 1994, Methods Mol. Biol., 28:253 260; and Sambrook et al., 2000, Molecular Cloning - A Laboratory Manual, Third Edition, CSH Laboratories). As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions. The terminology "amplification pair" or "primer pair" refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes.

[0052] As used herein, the terms "hybridizing" and "hybridizes" are intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 60%, at least about 70%, at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other. A preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 1X SSC, 0.1 % SDS at 50°C, preferably at 55°C, preferably at 60°C and even more preferably at 65°C. Highly stringent conditions include, for example, hybridizing at 68°C in 5x SSC/5x Denhardt's solution / 1.0% SDS and washing in 0.2x SSC/0.1 % SDS at room temperature. Alternatively, washing may be performed at 42°C. The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., supra; and Ausubel et al., supra (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.). Of course, a polynucleotide which hybridizes only to a poly (A) sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

[0053] The terms "identity" and "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e., overlapping positions) x 100). Preferably, the two sequences are the same length. Thus, In accordance with the present invention, the term "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, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70- 95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably, the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art. Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. Moreover, the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid. The present invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the herein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s). The skilled person will appreciate that all these different algorithms or programs will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[0054] In a related manner, the terms "homology" or "percent homology", refer to a similarity between two polypeptide sequences, but take into account changes between amino acids (whether conservative or not). As well known in the art, amino acids can be classified by charge, hydrophobicity, size, etc. It is also well known in the art that amino acid changes can be conservative (e.g., they do not significantly affect, or not at all, the function of the protein). A multitude of conservative changes are known in the art, Serine for threonine, isoleucine for leucine, arginine for lysine etc., Thus the term homology introduces evolutionistic notions (e.g., pressure from evolution to a retain function of essential or important regions of a sequence, while enabling a certain drift of less important regions).

[0055] The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[0056] In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. In another embodiment, the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:1 1-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available at the ALIGN Query using sequence data of the Genestream server IGH Montpellier France http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0057] The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the N BLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

[0058] By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more residues (including abasic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions. Contiguous bases of an oligomer are preferably at least about 80% (81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook ef al., Molecular Cloning, A Laboratory Manual, 2 ^nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91 , 7.37-7.57, 9.47-9.51 and 11.47-1 1.57, particularly at §§ 9.50-9.51 , 11.12- 1 1.13, 1 1.45-1 1.47 and 1 1.55-1 1.57).

[0059] The present invention refers to a number of units or percentages that are often listed in sequences. For example, when referring to "at least 80%, at least 85%, at least 90%...", or "at least about 80%, at least about 85%, at least about 90%...", every single unit is not listed, for the sake of brevity. For example, some units (e.g., 81 , 82, 83, 84, 85,... 91 , 92%....) may not have been specifically recited but are considered encompassed by the present invention. The non-listing of such specific units should thus be considered as within the scope of the present invention.

[0060] Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Patent Nos. 5,503,980 (Cantor), 5,202,231 (Drmanac et al.), 5,149,625 (Church et al.), 5,1 12,736 (Caldwell et al.), 5,068,176 (Vijg et al.), and 5,002,867 (Macevicz)). Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ one nucleotide (see U.S. Patent Nos. 5,837,832 and 5,861 ,242 (Chee et al.)).

[0061] A detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to an oligonucleotide probe. The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g., protein detection by far western technology: Guichet et al., 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001 , EMBO 20(3): 510-519). Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to use a detection method which is amenable to automation. A non-limiting example thereof includes a chip or other support comprising one or more (e.g., an array) of different probes.

[0062] A "label" refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified sequence) or to a polypeptide to be detected. Direct labeling can occur through bonds or interactions that link the label to the polynucleotide or polypeptide (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker" or bridging moiety, such as additional nucleotides, amino acids or other chemical groups, which are either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound).

[0063] As used herein, "expression" is meant the process by which a gene or otherwise nucleic acid sequence eventually produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s).

[0064] The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context required to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxyl terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al., supra. Sequence Listings programs can convert easily this one-letter code of amino acids sequence into a three-letter code.

[0065] The phrase "mature polypeptide" is defined herein as a polypeptide having biological activity a polypeptide of the present invention that is in its final form, following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, removal of signal sequences, glycosylation, phosphorylation, etc. In one embodiment, polypeptides of the present invention comprise mature of polypeptides of any one of the polypeptides disclosed herein. Mature polypeptides of the present invention can be predicted using programs such as SignalP. The phrase "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide as defined above. As well known, some nucleotide sequences are non-coding.

[0066] As used herein, the term "purified" or "isolated" refers to a molecule (e.g., polynucleotide or polypeptide) having been separated from a component of the composition in which it was originally present. Thus, for example, an "isolated polynucleotide" or "isolated polypeptide" has been purified to a level not found in nature. A "substantially pure" molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. For the sake of brevity, the units (e.g., 66, 67...81 , 82, 83, 84, 85,...91 , 92%....) have not been specifically recited but are considered nevertheless within the scope of the present invention.

[0067] An "isolated polynucleotide" or "isolated nucleic acid molecule" is a nucleic acid molecule (DNA or RNA) that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

[0068] As used herein, an "isolated polypeptide" or "isolated protein" is intended to include a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0069] The term "variant" refers herein to a polypeptide, which is substantially similar in structure (e.g., amino acid sequence) to a polypeptide disclosed herein or encoded by a nucleic acid sequence disclosed herein without being identical thereto. Thus, two molecules can be considered as variants even though their primary, secondary, tertiary or quaternary structures are not identical. A variant can comprise an insertion, substitution, or deletion of one or more amino acids as compared to its corresponding native protein. A variant can comprise additional modifications (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc). As used herein, the term "functional variant" is intended to include a variant which is sufficiently similar in both structure and function to a polypeptide disclosed herein or encoded by a nucleic acid sequence disclosed herein, to maintain at least one of its native biological activities.

[0070] As used herein, the term "biomass" refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste or a combination thereof. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, and animal manure or a combination thereof. Biomass that is useful for the invention may include biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle. In one embodiment of the present invention, biomass that is useful includes corn cobs, corn stover, sawdust, and sugar cane bagasse.

[0071] As used herein, the terms "cellulosic" or "cellulose-containing material" refers to a composition comprising cellulose. As used herein, the term "lignocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemi-cellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

[0072] Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulose-containing material can be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. The cellulose-containing material can be any type of biomass including, but not limited to, wood resources, municipal solid waste, wastepaper, crops, and crop residues (e.g., see Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman. 1994. Bioresource Technology 50: 3-16; Lynd. 1990. Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65. pp.23-40. Springer-Verlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.

[0073] The phrase "cellulolytic enhancing activity" or "cellulolysis-enhancing" is defined herein as a biological activity which enhances the hydrolysis of a cellulose-containing material by proteins having cellulolytic activity. The term "cellulolytic activity" is defined herein as a biological activity which hydrolyzes a cellulose- containing material.

[0074] The phrase "lignocellulolytic enhancing activity" or "Ngnocellulolysis-enhancing" is defined herein as a biological activity which enhances the hydrolysis of a lignocellulose-containing material by proteins having lignocellulolytic activity. The term "lignocellulolytic activity" is defined herein as a biological activity which hydrolyzes a lignocellulose-containing material.

[0075] The term "thermostable", as used herein, refers to an enzyme that retains its function or protein activity at a temperature greater than 50°C; thus, a thermostable cellulose-degrading or cellulase-enhacing enzyme/protein retains the ability to degrade or enhace the degradation of cellulose at this elevated temperature. A protein or enzyme may have more than one enzymatic activity. For example, some polypeptide of the present invention exhibit bifunctional activities such as xylosidase/ arabinosidase activity. Such bifunctional enzymes may exhibit thermostability with regard to one activity, but not another, and still be considered as "thermostable".

BRIEF DESCRIPTION OF DRAWINGS

[0076] In the appended drawings:

[0077] Figure 1 is a schematic map of the pGBFIN-49 expression plasmid. [0078] Figures 2 and 3 show protein activity-temperature profiles of various secreted proteins from Amorphotheca resinae.

[0079] Figure 4 shows protein activity-temperature profiles of various secreted proteins from Rhizomucor pusillus.

[0080] In the appended Sequence Listing, SEQ ID NOs: 1-582 relate to sequences from Amorphotheca resinae; SEQ ID NOs: 583-1032 relate to sequences from Rhizomucor pusillus; and SEQ ID NOs: 1033-1542 relate to sequences from Calcarisporiella thermophila.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS POLYPEPTIDES OF THE INVENTION

[0081] In one aspect, the present invention relates to isolated polypeptides secreted by Amorphotheca resinae, Rhizomucor pusillus, or Calcarisporiella thermophila (e.g., Amorphotheca resinae strain DAOM194228, Rhizomucor pusillus strain CBS 183.67, or Calcarisporiella thermophila strain CBS 279.70) having an activity relating to the processing or degradation of biomass (e.g., cell wall deconstruction).

[0082] In another aspect, the present invention relates to isolated polypeptides comprising the amino acid sequences shown in any one of SEQ ID NOs: 389-582, 883-1032, and 1373-1542.

[0083] In another aspect, the present invention relates to isolated polypeptides sharing a minimum threshold of amino acid sequence identity with any one of the above-mentioned polypeptides. In specific embodiments, the present invention relates to isolated polypeptides having at least 60%, 65%, 70%, 71 %, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of the above-mentioned polypeptides. Other specific percentage units that have not been specifically recited here for brevity are nevertheless considered within the scope of the present invention.

[0084] In another aspect, the present invention relates to a polypeptide encoded by a polynucleotide of the present invention, which includes genomic (e.g., SEQ ID NOs: 1-194, 583-732, and 1033-1202), and coding (e.g., SEQ ID NOs: 195-388, 733-882, and 1203-1372) nucleic acid sequences disclosed herein, polynucleotides hybridizing under medium-high, high, or very high stringency conditions with a full-length complement thereof, as well as polynucleotides sharing a certain degree of nucleic acid sequence identity therewith.

[0085] In another aspect, the present invention relates to a polypeptide comprising an amino acid sequence encoded by at least one exonic nucleic acid sequence of any one of the genomic sequences corresponding to SEQ ID NOs: 1-194, 583-732, and 1033-1202 (e.g., the intron or exon segments defined by the exon boundaries listed in Tables 2A-2C) or a functional part thereof.

[0086] In another aspect, the present invention relates to functional variants of any one of the above- mentioned polypeptides. In another embodiment, the term "functional" or "biologically active" relates to the native enzymatic (e.g., catalytic) activity of a polypeptide of the present invention. In some embodiments, the present invention relates to a polypeptide comprising a biological activity of any one of the enzymes described below, or a polynucleotide encoding same.

[0087] "Carbohydrase" refers to any protein that catalyzes the hydrolysis of carbohydrates. "Glycoside hydrolase", "glycosyl hydrolase" or "glycosidase" refers to a protein that catalyzes the hydrolysis of the glycosidic bonds between carbohydrates or between a carbohydrate and a non-carbohydrate residue. Endoglucanases, cellobiohydrolases, beta-glucosidases, a-glucosidases, xylanases, beta-xylosidases, alpha-xylosidases, galactanases, a-galactosidases, beta-galactosidases, a-amylases, glucoamylases, endo-arabinases, arabinofuranosidases, mannanases, beta-mannosidases, pectinases, acetyl xylan esterases, acetyl mannan esterases, femlic acid esterases, coumaric acid esterases, pectin methyl esterases, and chitosanases are examples of glycosidases.

[0088] "Cellulase" refers to a protein that catalyzes the hydrolysis of 1 ,4-D-glycosidic linkages in cellulose (such as bacterial cellulose, cotton, filter paper, phosphoric acid swollen cellulose, Avicel®); cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose); plant lignocellulosic materials, beta-D-glucans or xyloglucans. Cellulose is a linear beta-(1 ) glucan consisting of anhydrocellobiose units. Endoglucanases, cellobiohydrolases, and beta- glucosidases are examples of cellulases.

[0089] "Endoglucanase" refers to a protein that catalyzes the hydrolysis of cellulose to oligosaccharide chains at random locations by means of an endoglucanase activity.

[0090] "Cellobiohydrolase" refers to a protein that catalyzes the hydrolysis of cellulose to cellobiose via an exoglucanase activity, sequentially releasing molecules of cellobiose from the reducing or non-reducing ends of cellulose or cello- oligosaccharides. "Beta-glucosidase" refers to an enzyme that catalyzes the conversion of cellobiose and oligosaccharides to glucose.

[0091] "Hemicellulase" refers to a protein that catalyzes the hydrolysis of hemicellulose, such as that found in lignocellulosic materials. Hemicelluloses are complex polymers, and their composition often varies widely from organism to organism, and from one tissue type to another. Hemicelluloses include a variety of compounds, such as xylans, arabinoxylans, xyloglucans, mammans, glucomannans, and galacto(gluco)mannans. Hemicellulose can also contain glucan, which is a general term for beta-linked glucose residues. In general, a main component of hemicellulose is beta-1 ,4-linked xylose, a five carbon sugar. However, this xylose is often branched as beta-1 ,3 linkages or beta-1 , 2 linkages, and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, or by esterification to acetic acid. Hemicellulolytic enzymes, i.e., hemicellulases, include both endo-acting and exo-acting enzymes, such as xylanases, beta-xylosidases, alpha- xylosidases, galactanases, a-galactosidases, beta-galactosidases, endo-arabinases, arabinofuranosidases, mannanases, and beta-mannosidases. Hemicellulases also include the accessory enzymes, such as acetylesterases, ferulic acid esterases, and coumaric acid esterases. Among these, xylanases and acetyl xylan esterases cleave the xylan and acetyl side chains of xylan and the remaining xylo-oligomers are unsubstituted and can thus be hydrolysed with beta-xylosidase only. In addition, several less known side activities have been found in enzyme preparations which hydrolyze hemicellulose. Accordingly, xylanases, acetylesterases and beta- xylosidases are examples of hemicellulases. [0092] "Xylanase" specifically refers to an enzyme that hydrolyzes the beta-1 ,4 bond in the xylan backbone, producing short xylooligosaccharides.

[0093] "Beta-mannanase" or "endo-1,4-beta-mannosidase" refers to a protein that hydrolyzes mannan- based hemicelluloses (mannan, glucomannan, galacto(gluco)mannan) and produces short beta-1 , 4- mannooligosaccharides.

[0094] "Mannan endo-1,6-alpha-mannosidase" refers to a protein that hydrolyzes 1 ,6-alpha-mannosidic linkages in unbranched 1 ,6-mannans.

[0095] "Beta-mannosidase" (beta-1 ,4-mannoside mannohydrolase; EC 3.2.1.25) refers to a protein that catalyzes the removal of beta-D-mannose residues from the non-reducing ends of oligosaccharides.

[0096] "Galactanase", "endo-beta-1,6-galactanse" or "arabinogalactan endo-1,4-beta-galactosidase" refers to a protein that catalyzes the hydrolysis of endo-1 ,4-beta-D-galactosidic linkages in arabinogalactans.

[0097] "Glucoamylase" refers to a protein that catalyzes the hydrolysis of terminal 1 ,4-linked-D-glucose residues successively from non-reducing ends of the glycosyl chains in starch with the release of beta-D- glucose.

[0098] "Beta-hexosaminidase" or "beta-N-acetylglucosaminidase" refers to a protein that catalyzes the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosamines.

[0099] "Alpha-L-arabinofuranosidase", "alpha-N-arabmofuranosidase", "alpha-arabinofuranosidase", "arabinosidase" or "arabinofuranosidase" refers to a protein that hydrolyzes arabinofuranosyl-containing hemicelluloses or pectins. Some of these enzymes remove arabinofuranoside residues from 0-2 or 0-3 single substituted xylose residues, as well as from 0-2 and/or 0-3 double substituted xylose residues. Some of these enzymes remove arabinose residues from arabinan oligomers.

[00100] "Endo-arabinase" refers to a protein that catalyzes the hydrolysis of 1 ,5-alpha-arabinofuranosidic linkages in 1 ,5-arabinans.

[00101] "Exo-arabinase" refers to a protein that catalyzes the hydrolysis of 1 ,5-alpha-linkages in 1 ,5- arabinans or 1 ,5-alpha-L arabino-oligosaccharides, releasing mainly arabinobiose, although a small amount of arabinotriose can also be liberated.

[00102] "Beta-xylosidase" refers to a protein that hydrolyzes short 1 ,4-beta-D-xylooligomers into xylose.

[00103] "Cellobiose dehydrogenase" refers to a protein that oxidizes cellobiose to cellobionolactone.

[00104] "Chitosanase" refers to a protein that catalyzes the endohydrolysis of beta-1 ,4-linkages between D-glucosamine residues in acetylated chitosan (i.e., deacetylated chitin).

[00105] "Exo-polygalacturonase" refers to a protein that catalyzes the hydrolysis of terminal alpha 1 ,4- linked galacturonic acid residues from non-reducing ends thus converting polygalacturonides to galacturonic acid.

[00106] "Acetyl xylan esterase" refers to a protein that catalyzes the removal of the acetyl groups from xylose residues. "Acetyl mannan esterase" refers to a protein that catalyzes the removal of the acetyl groups from mannose residues, "ferulic esterase" or "ferulic acid esterase" refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and ferulic acid. "Coumaric acid esterase" refers to a protein that hydrolyzes the ester bond between the arabinose substituent group and coumaric acid. Acetyl xylan esterases, ferulic acid esterases and pectin methyl esterases are examples of carbohydrate esterases.

[00107] "Pectate lyase" and "pectin lyases" refer to proteins that catalyze the cleavage of 1 ,4-alpha-D- galacturonan by beta-elimination acting on polymeric and/or oligosaccharide substrates (pectates and pectins, respectively).

[00108] "Endo-1 ,3-beta-glucanase" or "laminarinase" refers to a protein that catalyzes the cleavage of 1 ,3-linkages in beta-D-glucans such as laminarin or lichenin. Laminarin is a linear polysaccharide made up of beta-1 ,3-glucan with beta-1 ,6-linkages.

[00109] "Lichenase" refers to a protein that catalyzes the hydrolysis of lichenan, a linear, 1 ,3-1 ,4-beta-D glucan.

[00110] Rhamnogalacturonan is composed of alternating alpha-1 ,4-rhamnose and alpha-1 ,2-linked galacturonic acid, with side chains linked 1 ,4 to rhamnose. The side chains include Type I galactan, which is beta-1 , 4-linked galactose with alpha-1 , 3-linked arabinose substituents; Type II galactan, which is beta-1 ,3-1 ,6- linked galactoses (very branched) with arabinose substituents; and arabinan, which is alpha-1 , 5-linked arabinose with alpha-1 , 3-linked arabinose branches. The galacturonic acid substituents may be acetylated and/or methylated.

[00111] "Exo-rhamnogalacturonanase" refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin from the non-reducing end.

[00112] "Rhamnogalacturonan acetylesterase" refers to a protein that catalyzes the removal of the acetyl groups ester-linked to the highly branched rhamnogalacturonan (hairy) regions of pectin.

[00113] "Rhamnogalacturonan lyase" refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin via a beta-elimination mechanism (e.g., see Pages et al., J. Bacteriol., 185:47274733 (2003)).

[00114] "Alpha-rhamnosidase" refers to a protein that catalyzes the hydrolysis of terminal non-reducing alpha-L-rhamnose residues in alpha-L-rhamnosides.

[00115] Certain proteins of the present invention may be classified as "Family 61 glycosidases" based on homology of the polypeptides to CAZy Family GH61. Family 61 glycosidases may exhibit cellulolytic enhancing activity or endoglucanase activity. Additional information on the properties of Family 61 glycosidases may be found in U.S. Patent Application Publication Nos. 2005/0191736, 2006/0005279, 2007/0077630, and in PCT Publication No.. WO 2004/031378.

[00116] "Esterases" represent a category of various enzymes including lipases, phospholipases, cutinases, and phytases that catalyze the hydrolysis and synthesis of ester bonds in compounds.

[00117] The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes where each enzyme is described by a sequence of four numbers preceded by "EC". The first number broadly classifies the enzyme based on its mechanism. According to the naming conventions, enzymes are generally classified into six main family classes and many sub-family classes: EC 1 Oxidoreductases: catalyze oxidation/reduction reactions; EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group); EC 3 Hydrolases: catalyze the hydrolysis of various bonds; EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation; EC 5 Isomerases: catalyze isomerization changes within a single molecule; and EC 6 Ligases: join two molecules with covalent bonds. A number of bioinformatic tools are available to the skilled person to predict which main family class and sub-family class an enzyme molecule belongs to according to its sequence information. In some instances, certain enzymes (or family of enzymes) can be re-classified, for example, to take into account newly discovered enzyme functions or properties. Accordingly, the polypeptides/enzymes of the present invention are not meant to be limited to specific enzyme classes as they currently exist. The skilled person would know how to appropriately reclassify (and assign the appropriate functions) to the enzymes of the present invention based on the amino acid sequence information provided herein. Such reclassifications are thus within the scope of the present invention.

[00118] In some embodiments, the present invention relates to a polypeptide comprising a biological activity of any one of the enzymes (or sub-classes thereof), or a polynucleotide encoding same.

• Cellulose-hydrolyzing enzymes, including: endoglucanases (EC 3.2.1.4), which hydrolyze the beta-1 ,4- linkages between glucose units; exoglucanases (also known as cellobiohydrolases 1 and 2) (EC 3.2.1.91), which hydrolyze cellobiose, a glucose disaccharide, from the reducing and non-reducing ends of cellulose; and beta-glucosidases (EC 3.2.1.21), which hydrolyze the beta-1 ,4 glycoside bond of cellobiose to glucose;

• Proteins that enhance or accelerate the action of cellulose-degrading enzymes, including: glycoside hydrolase family 61 (GH61), recently reclassified as AA9, proteins (e.g., polysaccharide monooxygenases), which enhance the action of cellulose enzymes on lignocellulose substrates;

• Enzymes that degrade or modify xylan and/or xylan-lignin complexes, including: xylanases, such as endo-1,4-beta-xylanase (EC 3.2.1.8), which catalyze the endohydrolysis of 1 -beta-D-xylosidic linkages in xylans (or xyloglucans); xylosidases, such as xylan 1,4-beta-xylosidases (EC 3.2.1.37), which catalyze hydrolysis of 1 ,4-beta-D-xylans to remove successive D-xylose residues from the non- reducing terminals, and also cleaves xylobiose; arabinosidases, such as alpha- arabinofuranosidases (EC 3.2.1.55), which hydrolyze terminal non-reducing alpha-L- arabinofuranoside residues in alpha-L-arabinosides (including arabinoxylans and arabinogalactans); alpha-glucuronidases (EC 3.2.1.139), which hydrolyze an alpha-D-glucuronoside to the corresponding alcohol and D-glucuronate; feruloyl esterases (EC 3.1.1.73), which catalyzes hydrolysis of the 4- hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar (which is usually arabinose in natural substrates); and acetylxylan esterases (EC 3.1.1.72), which catalyze deacetylation of xylans and xylo-oligosaccharides;

• Enzymes that degrade or modify mannan, including: mannanases, such as mannan endo-1,4-beta- mannosidase (EC 3.2.1.78), which catalyze random hydrolysis of 1 ,4-beta-D-mannosidic linkages in mannans, galactomannans and glucomannans; mannosidases (EC 3.2.1.25), which hydrolyze terminal, non-reducing beta-D-mannose residues in beta-D-mannosides; alpha-galactosidases (EC 3.2.1.22), which hydrolyzes terminal, non-reducing alpha-D-galactose residues in alpha-D-galactosides (including galactose oligosaccharides, galactomannans and galactohydrolase); and mannan acetyl esterases;

Enzymes that degrade or modify xyloglucans, including: xyloglucanases such as xyloglucan-specific endo-beta-1 ,4-glucanase (EC 3.2.1.151), which involves endohydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan; and xyloglucan-specific exo-beta-1 ,4-glucanase (EC 3.2.1.155), which catalyzes exohydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan; endoglucanases / cellulases;

Enzymes that degrade or modify glucans, including: Enzymes that degrade beta-1 ,4-glucan, such as endoglucanases; cellobiohydrolases; and beta-glucosidases;

Enzymes that degrade beta-1 , 3-1 ,4-glucan, such as endo-beta-1, 3(4)-glucanases (EC 3.2.1.6), which catalyzes endohydrolysis of 1 ,3- or 1 ,4-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3; endoglucanases (beta-glucanase, cellulase), and beta-glucosidases;

Enzymes that degrade or modify galactans, including: galactanases (EC 3.2.1.23), which hydrolyze terminal non-reducing beta-D-galactose residues in beta-D-galactosides;

Enzymes that degrade or modify arabinans, including: arabinanases (EC 3.2.1.99), which catalyze endohydrolysis of 1 ,5-alpha-arabinofuranosidic linkages in 1 ,5-arabinans;

Enzymes that degrade or modify starch, including: amylases, such as alpha-amylases (EC 3.2.1.1), which catalyze endohydrolysis of 1 ,4-alpha-D-glucosidic linkages in polysaccharides containing three or more 1 ,4-alpha-linked D-glucose units; and glucosidases, such as alpha-glucosidases (EC 3.2.1.20), which hydrolyze terminal, non-reducing 1 ,4-linked alpha-D-glucose residues with release of alpha-D- glucose;

Enzymes that degrade or modify pectin, including: pectate lyases (EC 4.2.2.2), which carry out eliminative cleavage of pectate to give oligosaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends; pectin lyases (EC 4.2.2.10), which catalyze eliminative cleavage of (14)- alpha-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-0-methyl-alpha-D-galact-4- enuronosyl groups at their non-reducing ends; polygalacturonases (EC 3.2.1.15), which carry out random hydrolysis of 1 ,4-alpha-D-galactosiduronic linkages in pectate and other galacturonans; pectin esterases, such as pectin acetyl esterase (EC 3.1.1.11 ), which hydrolyzes acetate from pectin acetyl esters; alpha-arabinofuranosidases; beta-galactosidases; galactanases; arabinanases; rhamnogalacturonases (EC 3.2.1.-), which hydrolyze alpha-D-galacturonopyranosyl-(1 ,2)-alpha-L- rhamnopyranosyl linkages in the backbone of the hairy regions of pectins; rhamnogalacturonan lyases (EC 4.2.2.-), which degrade type I rhamnogalacturonan from plant cell walls and releases disaccharide products; rhamnogalacturonan acetyl esterases (EC 3.1.1.-), which hydrolyze acetate from rhamnogalacturonan; and xylogalacturonosidases and xylogalacturonases (EC 3.2.1.-), which hydrolyze xylogalacturonan (xga), a galacturonan backbone heavily substituted with xylose, and which is one important component of the hairy regions of pectin;

• Enzymes that degrade or modify lignin, including: lignin peroxidases (EC 1.11.1.14), which oxidize lignin and lignin model compounds using hydrogen peroxide; manganese-dependent peroxidases (EC 1.1 1.1.13), which oxidizes lignin and lignin model compounds using Mn ²⁺ and hydrogen peroxide; versatile peroxidases (EC 1.1 1.1.16), which oxidize lignin and lignin model compounds using an electron donor and hydrogen peroxide and combines the substrate-specificity characteristics of the two other ligninolytic peroxidases: manganese peroxidase (EC 1.1 1.1.13) and lignin peroxidase (EC 1.11.1.14); and laccases (EC 1.10.3.2), a group of multi-copper proteins of low specificity acting on both o- and p-quinols, and often acting also on lignin; and

• Enzymes acting on chitin, including: chitinases (EC 3.2.1.14), which catalyze random hydrolysis of N- acetyl-beta-D-glucosaminide 1 ,4-beta-linkages in chitin and chitodextrins; and hexosaminidases, such as beta-N-acetylhexosaminidase (EC 3.2.1.52), which hydrolyzes terminal non-reducing N-acetyl-D- hexosamine residues in N-acetyl-beta-D-hexosaminides.

[00119] In another embodiment, the present invention includes the polypeptides and their corresponding activities as defined in Tables 1A-1 C, as well as functional variants thereof.

[00120] As alluded to above, the term "functional variant" as used herein is intended to include a polypeptide which is sufficiently similar in structure and function to any one of the above-mentioned polypeptides (without being identical thereto) to maintain at least one of its native biological activities. In another embodiment, a functional variant can comprise an insertion, substitution, or deletion of one or more amino acids as compared to its corresponding native protein. In another embodiment, a functional variant can comprise additional modifications (e.g., post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc).

[00121] In another embodiment, functional variants of the present invention can contain one or more conservative substitutions of a polypeptide sequence disclosed herein. Such modifications can be carried out routinely using site-specific mutagenesis. The term "conservative substitution" is intended to indicate a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acids having similar side chains are known in the art and include amino acids with basic side chains (e.g., lysine, arginine and hystidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

[00122] In another embodiment, functional variants of the present invention can contain one or more insertions, deletions or truncations of non-essential amino acids. As used herein, a "non-essential amino acid" is a residue that can be altered in a polypeptide of the present invention without substantially altering its (biological) function or protein activity. For example, amino acid residues that are conserved among the proteins of the present invention having similar biological activities (and their orthologs) are predicted to be particularly unamenable to alteration.

[00123] In another embodiment, functional variants can include functional fragments (i.e., biologically active fragments) of any one of the polypeptide sequences disclosed herein. Such fragments include fewer amino acids than the full length protein from which they are derived, but exhibit at least one biological activity of the corresponding full-length protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the full-length protein. A biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the present invention.

[00124] In another embodiment, the present invention includes other functional variants of the polypeptides disclosed herein, which can be identified by techniques known in the art. For example, functional variants can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants), of polypeptides of the present invention for biological activity. In another embodiment, a variegated library of variants can be generated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the present invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (e.g., see Narang (1983) Tetrahedron 39:3; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 1 1 :477).

[00125] In addition, libraries of fragments of the coding sequence of a polypeptide of the present invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.

[00126] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of polypeptides of the present invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:781 1-7815; Delgrave et al., (1993) Protein Engineering 6(3): 327-331 ).

[00127] In another embodiment, functional variants of the present invention can encompass orthologs of the genes and polypeptides disclosed herein. Orthologs of the polypeptides disclosed herein include proteins that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologs can be identified as comprising an amino acid sequence that is substantially homologous (shares a certain degree of amino acid sequence identity) with the polypeptides disclosed herein. As used herein, the expression "substantially homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain. For example, amino acid or nucleotide sequences which contain a common domain having at least 70%, 71 %, 72%, 73% 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 % 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity are defined herein as sufficiently identical.

[00128] In another embodiment, the present invention includes improved proteins derived from the polypeptides of the present invention. Improved proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the coding sequences of the polypeptides of the present invention such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of the resulting protein and thus improved proteins may be selected.

Recovery and purification

[00129] In another aspect, polypeptides of the present invention may be present alone (e.g., in an isolated or purified form), within a composition (e.g., an enzymatic composition for carrying out an industrial process), or in an appropriate host. In one embodiment, polypeptides of the present invention can be recovered and purified from cell cultures (e.g., recombinant cell cultures) by methods known in the art. In another embodiment, high performance liquid chromatography ("HPLC") can be employed for the purification.

[00130] In another aspect, polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending on the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Fusion proteins

[00131] In another aspect, the present invention includes fusion proteins comprising a polypeptide of the present invention or a functional variant thereof, which is operatively linked to one or more unrelated polypeptide (e.g., heterologous amino acid sequences). "Unrelated polypeptides" or "heterologous polypeptides" or "heterologous sequences" refer to polypeptides or sequences which are usually not present close to or fused to one of the polypeptides of the present invention. Such "unrelated polypeptides" or "heterologous polypeptides" having amino acid sequences corresponding to proteins which are not substantially homologous to the polypeptide sequences disclosed herein. Such "unrelated polypeptides" can be derived from the same or a different organism. In one embodiment, a fusion protein of the present invention comprises at least two biologically active portions or domains of polypeptide sequences disclosed herein. In the context of fusion proteins, the term "operatively linked" is intended to indicate that all of the different polypeptides are fused in- frame to each other. In another embodiment, an unrelated polypeptide can be fused to the N terminus or C terminus of a polypeptide of the present invention.

[00132] In another embodiment, a polypeptide of the present invention can be fused to a protein which enables or facilitates recombinant protein purification and/or detection. For example, a polypeptide of the present invention can be fused to a protein such as glutathione S-transferase (GST), and the resulting fusion protein can then be purified/detected through the high affinity of GST for glutathione.

[00133] Fusion proteins of the present invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences can be ligated together in frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (e.g., see Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the present invention can be cloned into such an expression vector so that the fusion moiety is linked in-frame to the polypeptide of interest.

Signal sequences

[00134] In another embodiment, a polypeptide of the present invention can be fused to a heterologous signal sequence (e.g., at its N terminus) to facilitate its isolation, expression and/or secretion from certain host cells (e.g., mammalian and yeast host cells). Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides may contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.

[00135] For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

[00136] The signal sequence can direct secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by known methods. In another embodiment, a signal sequence can be linked to a fusion protein of the present invention to facilitate detection, purification, and/or recovery thereof. For example, the sequence encoding a fusion protein of the present invention may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide. In another embodiment, the marker sequence can be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821 -824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. In another embodiment, the HA tag is another peptide useful for purification, which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.

POLYNUCLEOTIDES

[00137] The nucleic acid sequences of the genes disclosed herein were determined by sequencing cDNA clones, mRNA transcripts, or genomic DNA obtained from Amorphotheca resinae strain DAOM194228, Rhizomucor pusillus strain CBS 183.67, and Calcarisporiella thermophila strain CBS 279.70.

[00138] In another aspect, the present invention relates to polynucleotides encoding a polypeptide of the present invention, including functional variants thereof. In one embodiment, polynucleotides of the present invention comprise the coding nucleic acid sequence of any one of SEQ ID NOs: 195-388, 733-882, and 1203-1372, or as set forth in Tables 1A-1 C.

[00139] In another aspect, the present invention relates to genomic DNA sequences corresponding to the above mentioned coding sequences. In one embodiment, polynucleotides of the present invention comprise the genomic nucleic acid sequence of any one of SEQ ID NOs: 1-194, 583-732, and 1033-1202; or as set forth in Tables 1A-1C.

[00140] In another aspect, the present invention relates to a polynucleotide comprising at least one intronic or exonic nucleic acid sequence of any one of the genomic sequences corresponding to SEQ ID NOs: 1-194, 583-732, and 1033-1202 (e.g., the intron or exon segments defined by the exon boundaries listed in Tables 2A- 2C). Although only the positions of the exons are defined in Tables 2A-2C, a person of skill in the art would readily be able to determine the positions of the corresponding introns in view of this information. In some embodiments, polynucleotides comprising at least one these intronic segments are within the scope of the present invention.

[00141] In yet another aspect, the present invention relates to a polynucleotide comprising at least one exonic nucleic acid sequence comprised within SEQ ID NOs: 1-194, 583-732, and 1033-1202, or as set forth in Tables 2A-2C.

[00142] In another aspect, the present invention relates to isolated polynucleotides sharing a minimum threshold of nucleic acid sequence identity with any one of the above-mentioned polynucleotides. In specific embodiments, the present invention relates to isolated polynucleotides having at least 60%, 65%, 70%, 71 %, 72, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity to any one of the above- mentioned polynucleotides. Other specific percentage units that have not been specifically recited here for brevity are nevertheless considered within the scope of the present invention. Polynucleotides having the aforementioned thresholds of nucleic acid sequence identity can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences of the present invention such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded polypeptide. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[00143] In another aspect, the present invention relates to a polynucleotide that hybridizes (or is hybridizable) under medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of any one of the polynucleotides defined above.

[00144] As used herein, "very low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.

[00145] As used herein, "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.

[00146] As used herein, "medium stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SOS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SOS at 55°C.

[00147] As used herein, "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.

[00148] As used herein, "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.

[00149] As used herein, "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.

[00150] In one embodiment, a polynucleotide of the present invention (or a fragment thereof) can be isolated using the sequence information provided herein in conjunction with standard molecular biology techniques (e.g., as described in Sambrook et al., supra. For example, suitable hybridization oligonucleotides (e.g., probes or primers) can be designed using all or a portion of the nucleic acid sequences disclosed herein and prepared by standard synthetic techniques (e.g., using an automated DNA synthesizer). The oligonucleotides can be employed in hybridization and/or amplification reactions, for example, to amplify a template of cDNA, mRNA or genomic DNA, according to standard PCR techniques. A polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.

[00151] In another aspect, the present invention relates to polynucleotides encoding functional variants of any one of the polypeptides of the present invention, including a biologically active fragment or domain thereof.

[00152] In another aspect, the present invention can include nucleic acid molecules (e.g., oligonucleotides) sufficient for use as primers and/or hybridization probes to amplify, sequence and/or identify nucleic acid molecules encoding a polypeptide of the present invention or fragments thereof. In some embodiments, the present invention relates to polynucleotides (e.g., oligonucleotides) that comprise, span, or hybridize specifically to exon-exon or exon-intron junctions of the genomic sequences identified herein, such as those defined in Tables 2A-2C. Designing such polynucleotides/oligonucleotides would be within the grasp of a person of skill in the art in view of the target sequence information disclosed herein and are thus encompassed by the present invention.

[00153] In another aspect, the present invention relates to polynucleotides comprising silent mutations or mutations that do not significantly alter the (biological) function or protein activity of the encoded polypeptide. Guidance concerning how to make phenotypically silent amino acid substitutions is provided for example in Bowie et al., Science 247:1306-1310 (1990) and in the references cited therein. Furthermore, it will be apparent for the skilled person that DNA sequence polymorphisms of the genes disclosed herein may exist within a given population, which may differ from the sequences disclosed herein. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Accordingly, in one embodiment, the present invention can include natural allelic variants and homologs of polynucleotides disclosed herein. [00154] In another aspect, polynucleotides of the present invention can comprise only a portion or a fragment of the nucleic acid sequences disclosed herein. Although such polynucleotides may not encode a functional polypeptide of the present invention, they are useful for example as probes or primers in hybridization or amplification reactions. Exemplary uses of such polynucleotides include: (1) isolating a gene (as allelic variant thereof) from cDNA library; (2) in situ hybridization (e.g., FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of mRNA corresponding to a polypeptide disclosed herein, or a homolog, ortholog or variant thereof, in specific tissues and/or cells; and (4) probes and primers that can be used as a diagnostic tool to analyze the presence of a nucleic acid hybridizable to a polynucleotide disclosed herein in a given biological (e.g., tissue) sample. It would be within the grasp of a skilled person to design specific oligonucleotides in view of the nucleic acid sequences disclosed herein. Oligonucleotides typically comprise a region of nucleotide sequence that hybridizes (preferably under highly stringent conditions) to at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 37, 39, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides of a polynucleotide of the present invention. In one embodiment, such oligonucleotides can be used for identifying and/or cloning other family members, as well as orthologs from other species. In another embodiment, the oligonucleotide can be attached to a detectable label (e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). Such oligonucleotides can also be used as part of a diagnostic method or kit for identifying cells which express a polypeptide of the present invention.

[00155] As would be understood by the skilled person, full-length complements of any one of the polynucleotides of the present invention are also encompassed. In one embodiment, the full-length complements are antisense molecules with respect to the coding strands of polynucleotides of the present invention, which hybridize (preferably under highly stringent conditions) to at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 37, 39, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides to a polynucleotide of the present invention.

Sequencing errors

[00156] The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the corresponding complete genes from the organism sequenced herein, which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.

[00157] Unless otherwise indicated, all nucleotide sequences disclosed herein were determined by sequencing using an automated DNA sequencer, and all amino acid sequences of polypeptides disclosed herein were predicted by translation based on the genetic code. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

[00158] The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct such errors.

VECTORS

[00159] Another aspect of the invention pertains to vectors (e.g., expression vectors), containing a polynucleotide encoding a polypeptide of the present invention.

[00160] As used herein, the term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[00161] In one embodiment, recombinant expression vectors of the invention can comprise a polynucleotide of the present invention in a form suitable for expression of the polynucleotide in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the present invention can be introduced into host cells to thereby produce proteins or peptides, encoded by polynucleotides as described herein (e.g., polypeptides of the present invention).

[00162] In another embodiment, recombinant expression vectors of the present invention can be designed for expression of polypeptides of the present invention in prokaryotic or eukaryotic cells. For example, these polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel supra). In another embodiment, recombinant expression vectors of the present invention can be transcribed and translated in vitro, for example using 17 promoter regulatory sequences and 17 polymerase.

[00163] In another embodiment, expression vectors of the present invention can include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

[00164] For expression, a DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled person. In a specific embodiment, promoters are preferred that are capable of directing a high expression level of biologically active polypeptides of the present invention (e.g., lignocellulose active proteins) from fungi. Such promoters are known in the art. The expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[00165] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al., (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.

[00166] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate. A polynucleotide encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide of the present invention, or on a separate vector. Cells stably transfected with a polynucleotide of the present invention can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[00167] Expression of proteins in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g., to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

[00168] Vectors preferred for use in bacteria are for example disclosed in WO-A1 -2004/074468. Other suitable vectors will be readily apparent to the skilled artisan. Known bacterial promoters suitable for use in the present invention include the promoters disclosed in WO-A1 -2004/074468.

[00169] As indicated, the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and antibiotic resistance (e.g., tetracyline or ampicillin) for culturing in E. coli and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium and certain Bacillus species; fungal cells such as Aspergillus species, for example A. niger, A. oryzae and A. nidulans, yeast cells such as Kluyveromyces, for example K. lactis and/or Pichia, for example P. pastoris; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

[00170] Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[00171] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signal may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals. In an embodiment, a polypeptide of the present invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification and/or detection.

HOST CELLS

[00172] In another aspect, the present invention features cells, e.g., transformed host cells or recombinant host cells that contain a polynucleotide or vector of the present invention. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced a polynucleotide or vector of the invention by means of recombinant DNA techniques. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, in particular the strain from which the polynucleotide and polypeptide sequences disclosed herein were derived.

[00173] In one embodiment, a cell of the present invention is typically not a wild-type strain or a naturally- occurring cell. Host cells of the present invention can include, but are not limited to: fungi (e.g., Aspergillus niger, Trichoderma reesii, Myceliophthora thermophila and Talaromyces emersonii); yeasts (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica and Pichia pastoris); bacteria (e.g., Escherichia coli and Bacillus sp.); and plants (e.g., Nicotiana benthamiana, Nicotiana tabacum and Medicago sativa).

[00174] In another embodiment, a polynucleotide (or a polynucleotide which is comprised within a vector) may be homologous or heterologous with respect to the cell into which it is introduced. In this context, a polynucleotide is homologous to a cell if the polynucleotide naturally occurs in that cell. A polynucleotide is heterologous to a cell if the polynucleotide does not naturally occur in that cell. Accordingly, in an embodiment, the present invention relates to a cell which comprises a heterologous or a homologous sequence corresponding to any one of the polynucleotides or polypeptides disclosed herein.

[00175] In another embodiment, a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art.

[00176] In another embodiment, host cells can also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines. If desired, a stably transfected cell line can produce the polypeptides of the present invention. A number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al., (supra).

[00177] In another embodiment, the present invention relates to methods of inhibiting the expression of a polypeptide of the present invention in a host cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule (or a molecule comprising region of double-strandedness), wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length. The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA (miRNAs) for inhibiting translation. The present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of any one of the coding sequences of the polypeptides disclosed herein of inhibiting expression of that polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). The dsRNAs of the present invention can be used in gene-silencing methods. In one aspect, the invention relates to methods to selectively degrade RNA using the dsRNAi's of the present invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can be used to generate a loss-of-function mutation in a cell, an organ or an oganism. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Patent No. 6,506,559; U.S. Patent No. 6,51 1 ,824; U.S. Patent No. 6,515,109; and U.S. Patent No. 6,489,127.ln some instances, new phylogenic analyses of fungal species have resulted in taxonomic reclassifications. For example, following their phylogenic studies reported in van den Brink et al., ("Phylogeny of the industrial relevant, thermophilic genera Myceliophthora and Corynascus", Fungal Diversity (2012), 52:197-207), the authors proposed renaming all existing Corynascus species to Myceliophthora. Such changes in taxonomic classification are within the scope of the present invention and, regardless of future reclassifications, a person of skill in the art would be able to identify the organism used to determine the sequences disclosed herein for example based on the strain's accession number (DAOM194228, CBS 183.67, or CBS 279.70).

[00178] It should be understood herein that the level of expression of polypeptides of the present invention could be modified by adapting the codon usage ratio of a sequence of the present invention to that of the host or hosts in which it is meant to be expressed. This adaptation and the concept of codon usage ratio are all well known in the art.

Antibodies

[00179] In another aspect, the present invention relates to an isolated binding agent capable of selectively binding to a polypeptide of the present invention. Suitable binding agents may be selected from an antibody, an antigen binding fragment, or a binding partner. In one embodiment, the binding agent selectively binds to an amino acid sequence selected from Tables 1A-1 C, including to any fragment of any of the above sequences comprising at least one antibody binding epitope.

[00180] According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).

[00181] Antibodies are characterized in that they comprise immunoglobulin domains and as such, they are members of the immunoglobulin superfamily of proteins. An antibody of the invention includes polyclonal and monoclonal antibodies, divalent and monovalent antibodies, bi- or multi-specific antibodies, serum containing such antibodies, antibodies that have been purified to varying degrees, and any functional equivalents of whole antibodies. Isolated antibodies of the present invention can include serum containing such antibodies, or antibodies that have been purified to varying degrees. Whole antibodies of the present invention can be polyclonal or monoclonal. Alternatively, functional equivalents of whole antibodies, such as antigen binding fragments in which one or more antibody domains are truncated or absent (e.g., Fv, Fab, Fab', or F(ab)2 fragments), as well as genetically-engineered antibodies or antigen binding fragments thereof, including single chain antibodies or antibodies that can bind to more than one epitope (e.g., bi-specific antibodies), or antibodies that can bind to one or more different antigens (e.g., bi- or multi-specific antibodies), may also be employed in the invention. Methods for the generation and production of antibodies are well known in the art.

[00182] Monoclonal antibodies may be produced according to the methodology of Kohler and Milstein (Nature 256:495-497, 1975). Non-antibody polypeptides, sometimes referred to as binding partners, may be designed to bind specifically to a protein of the invention. Examples of the design of such polypeptides, which possess a prescribed ligand specificity are given in Beste et al., (Proc. Nat'l Acad. Sci. 96:1898-1903, 1999). In one embodiment, a binding agent of the invention is immobilized on a substrate such as: artificial membranes, organic supports, biopolymer supports and inorganic supports such as for use in a screening assay.

[00183] In some embodiment, antibodies and binding agents specifically binding to polypeptides of the present invention may be produced and used even in absence of knowledge of the precise biological function and/or protein activity of the polypeptide. Such antibodies and binding agent may be useful, for example, as diagnostic, classification, and/or research tools.

COMPOSITIONS AND USES

[00184] In another aspect, the present invention relates to a composition comprising one or more polypeptides or polynucleotides of the present invention. In one embodiment, the compositions are enriched in such a polypeptide. The term "enriched" indicates that the biological activity (e.g., biomass degradation or processing) of the composition has been increased, e.g., with an enrichment factor of at least 1.1. The composition may comprise a polypeptide of the present invention as the major component, e.g., a mono- component composition. Alternatively, the composition may comprise multiple enzymatic activities (e.g., those described herein).

[00185] The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art. Examples are given below of preferred uses of the polypeptide compositions of the present invention. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

[00186] In another aspect, the present invention relates to the use of the polypeptides (e.g., enzymes) of the present invention a number of industrial and other processes. Despite the long term experience obtained with these processes, there remains a need for improved polypeptides and enzymes featuring one or more significant advantages over those presently used. Depending on the specific application, these advantages can include aspects such as lower production costs, higher specificity towards the substrate, greater synergies with existing enzymes, less antigenic effect, less undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better properties of the final product, and food grade or kosher aspects. In various embodiments, the present invention seeks to provide one or more of these advantages, or others.

Biomass processing or degradation

[00187] In another aspect, the polypeptides of the present invention may be used in new or improved methods for enzymatically degrading or converting plant cell wall polysaccharides from biomass into various useful products. In addition to cellulose and hemicellulose, plant cell walls contain associated pectins and lignins, the removal of which by enzymes of the current invention can improve accessibility to cellulases and hemicellulases, or which can themselves be converted to useful products. Therefore the polypeptides of the present invention may be used to degrade biomass or pretreated biomass to sugars. These sugars may be used as such or may be, for example, fermented into ethanol.

[00188] Usually, biomass must be subjected to pre-treatment in order to make the cellulose more accessible. Accordingly, in one embodiment, polypeptides of the present invention may be used in improved methods for the processing of pretreated biomass. Pretreatment technologies may involve chemical, physical, or biological treatments. Examples of pre-treatment technologies include but are not limited to: steam explosion; ammonia; acid hydrolysis; alkaline hydrolysis; solvent extraction; crushing; milling; etc.

[00189] One example of a product produced from biomass is bioethanol. Bioethanol is usually produced by the fermentation of glucose to ethanol by yeasts such as Saccharomyces cerevisiae: in addition to ethanol, other chemicals may be synthesized starting from glucose. Ethanol, today, is produced mostly from sugars or starches, obtained from sugar cane, fruits and grains. In contrast, cellulosic ethanol is obtained from cellulose, the main component of wood, straw and much of the plants. Sources of biomass for cellulosic ethanol production comprise agricultural residues (e.g., leftover crop materials from stalks, leaves, and husks of corn plants), forestry wastes (e.g., chips and sawdust from lumber mills, dead trees, and tree branches), energy crops (e.g., dedicated fast- growing trees and grasses such as switch grass), municipal solid waste (e.g., household garbage and paper products), food processing and other industrial wastes (e.g., black liquor, paper manufacturing by-products, etc.).

[00190] Plant biomass is a mixture of plant polysaccharides, including cellulose, hemicelluloses, and pectin, together with the structural polymer, lignin. Glucose is released from cellulose by the action of mixtures of enzymes, including: endoglucanases, exoglucanases (cellobiohydrolases 1 and 2) and beta-glucosidases. Efficient large-scale conversion of cellulosic materials by such mixtures may require the full complement of enzymes, and can be enhanced by the addition of enzymes that attack the other plant cell wall components (e.g., hemicelluloses, pectins, and lignins), as well as chemical linkages between these components. Hence, polypeptides of the present invention that are highly expressed, or have high specific activity, stability, or resistance to inhibitors may improve the efficiency of the process, and lower enzyme costs. It would be an advantage to the art to improve the degradation and conversion of plant cell wall polysaccharides by composing cellulase mixtures using cellulase enzymes with such properties. Furthermore, polypeptides of the present invention that are able to function at extremes of pH and temperature are desirable, both since improved enzyme robustness decreases costs, and because enzymes that function at high temperature will allow high processing temperatures under high substrate consistency conditions that decrease viscosity and thus improve yields.

[00191] Glycoside hydrolases from the family GH61 are known to stimulate the activity of cellulose cocktails on lignocellulosic substrates and are thus considered to exhibit cellulose-enhancing activity (Harris et al., Biochemistry 49, 3305 (2010)). Enhancement of cellulase cocktail efficiency by GH61 proteins of the present invention may contribute to lowering the costs of cellulase enzymes used for the production of glucose from plant cell biomass, as described above. GH61 (glycoside hydrolase family 61 or sometimes referred to as EGIV) proteins are oxygen-dependent polysaccharide monooxygenases (PMO's) according to the latest literature. Often in the literature, these proteins are mentioned as enhancing the action of cellulases on lignocellulose substrates. GH61 was originally classified as an endoglucanase, based on the measurement of very weak endo- 1 ,4- -d-glucanase activity in one family member. The term "GH61" as used herein, is to be understood as a family of enzymes, which share common conserved sequence portions and foldings, originally classified in family 61 (http://www.cazy.org/GH61.html) of the well-established CAZY GH classification system, and now reclassified by CAZY as family AA9 (http://www.cazy.org/AA9.html). GH61 is used herein as being part of the cellulolytic system elaborated by certain fungi to degrade cellulose.

[00192] Enzymatic hydrolysis of plant hemicellulose yields 5-carbon sugars that either may be fermented to ethanol by some species of yeast, or converted to other types of chemical products. Enzymatic deconstruction of hemicellulose is also known to improve the accessibility of plant cell wall cellulose to cellulase enzymes for the production of glucose from lignocellulosic materials. Hemicellulase enzymes of the present invention that enhance glucose production from lignocellulose would find utility in the bioethanol industry and in other process that rely on glucose or pentose streams from lignocellulose.

[00193] Lignin is composed of methoxylated phenyl-propane units linked by ether linkages and carbon- carbon bonds. The chemical composition of lignin may, depending on species, include guaiacyl, 4- hydroxyphenyl, and syringyl groups. Enzymatic modification of lignin by the polypeptides of the present invention can be used for the production of structural materials from plant biomass, or alternatively improve the accessibility of plant cellulose and hemicelluloses to cellulase enzymes for the release of glucose from biomass as described above. Enzymes that degrade the lignin component of lignocellulose include lignin peroxidases, manganese-dependent peroxidases, versatile peroxidases, and laccases (Vicuna et al., 2000, Molecular Biotechnology 14: 173-176; Broda et al., 1996, Molecular Microbiology 19: 923-932). In some embodiments, polypeptides of the present invention may also, in certain instances, be active in the decolorization of industrial dyes, and thus useful for the treatment and detoxification of chemical wastes.

[00194] In another embodiment, pectin-degrading polypeptides of the present invention can also enhance the action of cellulases on plant biomass by improving the accessibilty of cellulase to the cellulose component of lignocellulose.

[00195] In another embodiment, polypeptides of the present invention may also be useful in other applications for hydrolyzing non-starch polysaccharide (NSP).

[00196] In another embodiment, esterases of the present invention can be useful in the bioenergy industry such as for the production of biodiesel and hydrolysis of hemicellulose.

[00197] In another embodiment, the present invention relates to methods for degrading or converting a cellulose-containing material, comprising: treating the cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity.

[00198] In another embodiment, the present invention relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

Food product industry

[00199] In one embodiment, the present invention relates to methods for preparing a food product comprising incorporating into the food product an effective amount of a polypeptide of the present invention. This can improve one or more properties of the food product relative to a food product in which the polypeptide is not incorporated. The phrase "incorporated into the food product" is defined herein as adding a polypeptide of the present invention to the food product, to any ingredient from which the food product is to be made, and/or to any mixture of food ingredients from which the food product is to be made. In other words, a polypeptide of the present invention may be added in any step of the food product preparation and may be added in one, two or more steps. The polypeptide of the present invention is added to the ingredients of a food product which can then be treated by methods including cooking, boiling, drying, frying, steaming or baking as is known in the art.

[00200] At least in the context of food products, the term "effective amount" is defined herein as an amount of the polypeptide (e.g., enzyme) of the present invention that is sufficient for providing a measurable effect on at least one property of interest of the food product. The term "improved property" is defined herein as any property of a food product which is improved by the action of a polypeptide (e.g., enzyme) of the present invention relative to a food product in which the polypeptide is not incorporated. The improved property may be determined by comparison of a food product prepared with and without addition of a polypeptide of the present invention. Organoleptic qualities may be evaluated using procedures well established in the food industry, and may include, for example, the use of a panel of trained taste-testers.

[00201] The polypeptides of the present invention may be prepared in any form suitable for the use in question, e.g., in the form of a dry powder, agglomerated powder, or granulate, in particular a non-dusting granulate, liquid, in particular a stabilized liquid, or protected enzyme such as described in WO01/1 1974 and WO02/26044. Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying the enzyme according to the invention onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g., a salt (such as NaCI or sodium sulphate), sugar (such as sucrose or lactose), sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy. In an embodiment, the polypeptide of the present invention (and/or additional polypeptides/enzymes) may be contained in slow-release formulations. Methods for preparing slow-release formulations are well known in the art. Adding nutritionally acceptable stabilizers such as sugar, sugar alcohol, or another polyol, and/or lactic acid or another organic acid according to established methods may for instance, stabilize liquid enzyme preparations.

[00202] In another embodiment, polypeptides of the present invention may also be incorporated in yeast- comprising compositions such as disclosed in EP-A-0619947, EP-A-0659344 and WO02/49441.

[00203] In another embodiment, one or more additional polypeptides/enzymes may be incorporated into a food product of the present invention. The additional enzyme may be of any origin, including mammalian and plant, and preferably of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art. Enzymes may conveniently be produced in microorganisms. Microbial enzymes are available from a variety of sources; Bacillus species are a common source of bacterial enzymes, whereas fungal enzymes are commonly produced in Aspergillus species.

[00204] In specific embodiments, additional polypeptides/enzymes include starch degrading enzymes, xylanases, oxidizing enzymes, fatty material splitting enzymes, or protein-degrading, modifying or crosslinking enzymes. Starch degrading enzymes include endo-acting enzymes such as alpha-amylase, maltogenic amylase, pullulanase or other debranching enzymes, and exo-acting enzymes that cleave off glucose (amyloglucosidase), maltose (beta-amylase), maltotriose, maltotetraose and higher oligosaccharides. Suitable xylanases are for instance xylanases, pentosanases, hemicellulase, arabinofuranosidase, glucanase, cellulase, cellobiohydrolase, beta-glucosidase, and others. Oxidizing enzymes are for instance glucose oxidase, hexose oxidase, pyranose oxidase, sulfhydryl oxidase, lipoxygenase, laccase, polyphenol oxidases and others. Fatty material splitting enzymes are for instance triacylglycerol lipases, phospholipases (such as A1 , A2, B, C and D) and galactolipases. Protein degrading, modifying or crosslinking enzymes are for instance endo-acting proteases (serine proteases, metalloproteases, aspartyl proteases, thiol proteases), exo-acting peptidases that cleave off one amino acid, or dipeptide, tripeptide etceteras from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of the polypeptide chain, asparagines or glutamine deamidating enzymes such as deamidase and peptidoglutaminase or crosslinking enzymes such as transglutaminase.

[00205] In others embodiments, additional polypeptides/enzymes can include: amylases, such as alpha- amylase (which can be useful for providing sugars that are fermentable by yeast) or beta-amylase; cyclodextrin glucanotransferase; peptidase (e.g., an exopeptidase, which can be useful in flavour enhancement); transglutaminase; lipase, which can be useful for the modification of lipids present in the food or food constituents), phospholipase, cellulase, hemicellulase, protein disulfide isomerase, peroxidase, laccase, or an oxidase (e.g., glucose oxidase, hexose oxidase, aldose oxidase, pyranose oxidase, lipoxygenase or L-amino acid oxidase).

[00206] In other embodiment, esterases of the present invention have a number of applications in the food industry including, but not limited to, degumming vegetable oils; improving the production of bread (e.g., in situ production of emulsifiers); producing crackers, noodles, and pasta; enhancing flavor development of cheese, butter, and margarine; ripening cheese; removing wax; trans-esterification of flavors and cocoa butter substitutes; synthesizing structured lipids for infant formula and nutraceuticals; improving the polyunsaturated fatty acid content in fish oil; and aiding in digestion and releasing minerals in food processing.

[00207] When one or more additional enzyme activities are to be added in accordance with the methods of the present invention, these activities may be added separately or together with the polypeptide according to the invention.

Detergent industry

[00208] In another aspect, polypeptides of the present invention can be useful in the detergent industry, e.g., for removal of carbohydrate-based stains from soiled laundry. Enzymes are used in detergents in order to improve its efficacy to remove most types of dirt. In some embodiments, esterases such as lipases of the present invention are particularly useful for removing fats and lipids.

Feed industry

[00209] In another aspect, polypeptides of the present invention can be useful in the feed enzyme industry, e.g., for increasing nutritional quality, digestibility and/or absorption of animal feed.

[00210] Feed enzymes have an important role to play in current farming systems, as they can increase the digestibility of nutrients, leading to greater efficiency in the production of animal products such as meat and eggs. At the same time, they can play a role in minimizing the environmental impact of increased animal production. [00211] Non-starch polysaccharides (NSP) can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance.

[00212] Endoxylanases and phytases are the best-known feed-enzyme products. Phytase enzymes hydrolyse phytic acid and release inorganic phosphate, thereby avoiding the need to add inorganic phosphates to the diet and reducing phosphorus excretion. Addition of xylanases to feed has also been shown to have positive effects on animal growth. Adding specific nutrients to feed improves animal digestion and thereby reduces feed costs. A lot of feed additives are being currently used and new concepts are continuously developed. Use of specific enzymes like non-starch carbohydrate degrading enzymes could breakdown fiber, releasing energy as well as increasing the protein digestibility due to better accessibility of the protein when fiber gets broken down. In this way the feed cost could come down, as well as the protein levels in the feed also could be reduced.

[00213] Non-starch polysaccharides (NSPs) are also present in virtually all feed ingredients of plant origin. NSPs are poorly utilized and can, when solubilized, exert adverse effects on digestion. Exogenous enzymes can contribute to a better utilization of these NSPs and as a consequence reduce any anti-nutritional effects. Accordingly, in a particular embodiment, hemicellulases and other polysaccharide-active polypeptides/enzymes of the present invention can be used for this purpose in cereal-based diets for poultry and, to a lesser extent, for pigs and other species.

[00214] In some embodiments, esterases of the present invention are useful in the feed industry such as for reducing the amount of phosphate in feed.

Pulp and paper

[00215] In another embodiment, xylanases of the present invention can be useful in the pulp and paper industry, e.g., for prebleaching of kraft pulp. Xylanases have been found to be most effective for that purpose. Xylanases attract increasing scientific and commercial attention due to applications in the pulp and paper industry for removal of hemicellulose from dissolving pulps or for enhancement of the bleachability of pulp and, thus, reduction of the use of environmentally harmful bleaching chemicals. A similar application of xylanases for pulp prebleaching is an already well-established technology and has greatly stimulated research on hemicellulases in the past decade. Although lignin-active peroxidases of the present invention may also be active in modification of lignin and hence have bleaching properties, such enzymes are generally less attractive for bleaching due to the need to use and recycle expensive redox mediators.

[00216] In a related embodiment, polypeptides such as xylanases of the present invention can be used to pre-bleach pulp to reduce the amount of bleaching chemicals to obtain a given brightness. It is suggested that xylanase depolymerises xylan blocks and increases accessibility or helps liberation of residual lignin by releasing xylan-chromophore fragments. In addition to brownstock prior to bleaching, polypeptides such as xylanases of the present invention can save on bleaching chemicals. The enzymes hydrolyze surface xylans and are able to break linkages between hemicellulose and lignin. Other polypeptides (e.g., hemicellulase active enzymes) of the present invention which can break these linkages can function effectively in bleaching or pre-bleaching of pulp, and thus such uses are also within the scope of the present invention.

[00217] In some embodiments, esterases of the present invention are useful for the removal of triglycerides, steryl esters, resin acids, free fatty acids, and sterols (e.g., lipophilic wood extractives).

Other uses

[00218] In another embodiment, polypeptides such as xylanases of the present invention can be used in antibacterial formulations, as well as in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash.

[00219] Chitin is a beta-(1 ,4)-linked polymer of N-acetyl D-glucosamine (GlcNAc), found as a structural polysaccharide in fungal cell walls as well as in the exoskeleton of arthropods and the outer shell of crustaceans. Approximately 75% the total weight of shellfish, is considered waste, and a large proportion of the material making up the waste is chitin. Accordingly, in one embodiment, polypeptides such as chitin-degrading enzymes of the present invention are useful in the modification and degradation of chitin, allowing the production of chitin- derived material, such as chitooligosaccharides and N-acetyl D-glucosamine, from chitin waste. In another embodiment, polypeptides such as chitinase enzymes of the present invention can be useful as antifungal agents.

[00220] In another embodiment, polypeptides of the present invention can be used in the textile industry (e.g., for the treatment of textile substrates). More particularly, cellulases (e.g., endo-, exocellulases and cellobiohydrolases) have gained importance in the treatment of cellulose-containing fibers. During the washing of indigo-dyed denim textiles, enzymatic treatment by a polypeptide of the present invention is can be used in place of (or in addition to) a bleaching treatment to achieve a "used" look of jeans or other suitable fabrics. Polypeptides of the present invention can also improve the softness/feel of such fabrics. When used in textile detergent compositions, enzymes of the present invention can enhance cleaning ability or act as a softening agent. In another embodiment, polypeptides such as cellulases of the present invention can be used in combination with polymeric agents in processes for providing a localized variation in the color density of fibers.

[00221] In another embodiment, polypeptides of the present invention can be used in the waste treatment industry (e.g., for changing the characteristics of the waste to become more amenable to further treatment and/or for bio-conversion to value-added products). Polypeptides such as lipases, cellulases, amylases, and proteases of the present invention can be used in addition to microorganisms to break down polymeric substances like proteins, polysaccharides and lipids, thereby facilitating this process.

[00222] In another embodiment, polypeptides of the present invention can be used in industries such as biocatalysis; sewage treatment; cleaning up oil pollution; the synthesis of fragrances; and enhancing the recovery of oil (e.g., during drilling).

[00223] Other uses of the polynucleotides and polypeptides of the present invention would be apparent to a person of skill in the art in view of the sequences and biological activities disclosed herein. These other uses, even though not explicitly mentioned here, are nevertheless within the scope of the present invention. Diagnostic, classification and research tools

[00224] In another embodiment, the polynucleotides, polypeptides and antibodies of the present invention can be useful for diagnostic and classification tools. In this regard, it would be within the capacities of a person of skill in the art to search existing sequence databases and perform a phylogenic analysis based on the nucleic acid and amino acid sequences disclosed herein. Furthermore, designing hybridization probes or primers that are specific for a particular genus, species or strain (e.g., the genus, species, or strain from which the sequences disclosed herein were derived) would be within the grasp of a skilled person, in view of the sequence information disclosed herein. Similarly, a skilled person would be able to select an epitope of a polypeptide of the present invention which is specific for a particular genus, species or strain (e.g., the genus, species, or strain from which the sequences disclosed herein were derived) and generate an antibody or binding agent that binds specifically thereto.

[00225] Such tools are useful, for example, in diagnostic methods for detecting the presence or absence of a particular organism (e.g., the organism from which the sequences disclosed herein were derived) in a sample; as research tools (e.g., for designing and producing microarrays for studying fungal gene expression); for rapidly classifying an organism of interest based the detection of a sequence or polypeptide specific for that organism. The skilled person would recognize that knowledge of the precise (biological) function or protein activity of a polypeptide of the present invention is not absolutely necessary for the aforementioned tools to be useful for diagnostic, research, or classification purposes. Sequences that are particularly useful in this regard are the genomic, coding and amino acid sequences corresponding to the polypeptides of the present invention annotated as "unknown" in Tables 1A-1C (as well as their corresponding exons and introns defined in Tables 2A-2C, where available). These sequences show little sequence identity with those in the art and thus can be useful as markers for identifying the organisms from which the sequences of the present invention were derived. The skilled person would know how to search various sequence databases to design specific hybridization oligonucleotides (e.g., probes and primers), as well as produce antibodies specifically binds to the aforementioned sequences.

[00226] In some embodiments, the present invention relates to a method for identifying and/or classifying an organism (e.g., a fungal species) based on a biological sample, the method comprising detecting the presence or absence of any one of the polynucleotides or polypeptides of the present invention (e.g., those recited in the preceding paragraph) and determining that said organism is present or classifying said organism based on the presence of the polynucleotide or polypeptide. In some embodiments, the detecting step can be carried out using one or more oligonucleotides or antibodies of the present invention. In some embodiments, the detecting step can be carried out by performing an amplification and/or hybridization reaction.

[00227] In Tables 1A-1 C below, the skilled person will recognize that although the precise protein activity of a polypeptide of the present invention may not be known per se (e.g., in the case of proteins of the presence invention labelled as "unknown" in Tables 1A-1 C), the polypeptide may be nevertheless useful for carrying out an industrial process (e.g., cellulase-enhancing, cellulose-degrading, hemicellulose-degrading, cellulolysis- enhancing, lignocellulolysis-enhancing, and other biological functions listed in Tables 1A-1C). In this regard, proteins labelled herein as "unknown" comprise proteins whose precise enzymatic activities may not be deduceable from sequence comparisons, but that are nevertheless indentified as interesting targets for industrial applications for other reasons (e.g., their expression is induced by growth under certain coonditions such as in the presence of cellulostic and/or lignocellulostic biomass).

Table 1A. Biomass degrading genes and polypeptides of Amorphotheca resinae

In the above Table, an asterix (*) refers to genomic sequences of the present PCT application (SEQ ID NOs: 151 , 152, 153, 156, 160, 163, 166, and 171 ) that were not able to be mapped to the genome from transciptome data (coding and amino acid sequences). Accordingly, in the Sequence Lsting, the nucleic acid sequences for these genomic sequences have been assigned an arbitrary number of bases "n" as placeholders.

Table 1 B. Biomass degrading genes and polypeptides of Rhizomucor pusillus

Table 1C. Biomass degrading genes and polypeptides of Calcarisporiella thermophila

In the above Table, an asterix (*) refers to a genomic sequence of the present PCT application (SEQ ID NO: 1157) that was not able to be mapped to the genome from transciptome data (coding and amino acid sequence). Accordingly, in the Sequence Lsting, the nucleic acid sequence for this genomic sequences has been assigned an arbitrary number of bases "n" as placeholders.

Table 2A. List of genes of Amorphotheca resinae with reference to exon boundaries

Genomic Genomic

Gene ID sequence sequence Exon boundaries (nucleotide positions) and exons

(SEQ ID NO:) length

Amore2p4_000017 1 1062 1..295, 347..398, 462..1062

Amore2p4_000018 2 1996 1..172, 223..5Θ7, 617..954, 1033..1813, 1860..1996

Amore2p4_000138 3 1385 1..370, 421. 1385

Amore2p4_000145 4 2005 1..198, 248..2005

Amore2p4_000150 5 1995 1. 1995

Amore2p4_000307 6 1635 1..1635

Amore2p4_000373 7 1156 1..90, 139..874, 993 .1156

Amore2p4_000422 8 1049 1..276, 324..561 , 609..968, 1018..1049

Amore2p4_000458 9 5162 1.-129, 183..297, 348..2533, 3466..4706, 5060..5162

Amore2p4_000494 10 1097 1..237, 297..1097

Amore2p4_000599 11 975 1..975

Amore2p4_000698 12 1504 1..37, 122..279, 337..963, 1014..1201 , 1255..1504

Amore2p4_000713 13 1059 1..174, 309..416, 463..1059

Amore2p4_000867 14 3164 1..141 , 192..469, 517..1182, 1231..1302, 1376..3164

Amore2p4_000917 15 1551 1 .201 , 268. 1261 , 1370. 1551

Amore2p4_001004 16 1149 1..1149

Amore2p4_001019 17 500 1..302, 368..500

Amore2p4_001041 18 1429 1.708, 759..808, 865..1429

Amore2p4_001152 19 1198 1..964, 014..1198

Amore2p4_001349 20 1838 1..375, 630..1838

Amore2p4_001406 21 1349 1..353, 415..695, 751..1349

Amore2p4_001441 22 3234 1..276, 325..3234

Amore2p4_001468 23 1865 1..93, 157 .691 , 748. 1307, 1371 .1865

Amore2p4_001513 24 1057 1..404, 457..1057

Amore2p4_001553 25 836 1..174, 228..836

Amore2p4_001600 26 1451 1..221. 273..379, 430..1451

Amore2p4_001688 27 1834 1..444, 512..1834

1.78, 289..491 , 615..630, 687 .966, 1023. 1114, 1187..1462,

Amore2p4_001820 28 3685

1516..1541 , 1704..2086, 2165..2332, 2475..3685

Amore2p4_001883 29 1308 1..1308

Amore2p4_001995 30 1583 1..335, 386..484, 545..1583

1..496, 548..1200, 249 .1494, 1547 .1565, 1673 .1823,

Amore2p4_002014 31 1994

1913. 1994

Amore2p4_002297 32 2072 1..325, 382 .489, 541..1553, 1610..1668, 1721..2072

Amore2p4_002375 33 1120 1..82, 132..294, 349.706, 755..1120

Amore2p4_002415 34 1365 1..249, 306.734, 783..927, 1052..1365

1.73, 127..158, 208..283, 431..451 , 500..1006, 1057..1363,

Amore2p4_002445 35 1625

1425..1489, 1549..1625

Amore2p4_002666 36 1506 1..144, 197 .301, 358.749, 807..898, 947..1506

Amore2p4_002714 37 1797 1..259, 309..424, 475.736, 788..1279, 1340..1606, 1733..1797

1..55, 117 .169, 397..451 , 625..640, 694..810, 860..955,

Amore2p4_002814 38 2596

1010..1194, 1249..1343, 1402..1651 , 1706. 1926, 1979..2596

Amore2p4_003056 39 1617 1..65, 120..182, 399.733, 797. 1617 Amore2p4_006431 80 1577 1..97, 147..666, 715..1111 , 1173..1577

Amore2p4_006440 81 1834 1 .1365, 1415..1834

Amore2p4_006504 82 2419 1 .115, 162..359, 539..705, 851..1265, 1434..2419

Amore2p4_006606 83 1826 1..376, 424..550, 595..1156, 1227..1714, 1760..1826

Amore2p4_006655 84 2612 1..139, 194..276, 329. 413, 517..2612

Amore2p4_006713 85 2167 1..122, 169..470, 515..571 , 616. 1411 , 1459..2167

Amore2p4_006838 86 1826 1..262, 320..435, 487.748, 79Θ..892, 1021..1418, 1495..1826

Amore2p4_006842 87 885 1..885

Amore2p4_006872 88 1336 1..94, 156..1336

Amore2p4_006919 89 1338 1..359, 409..1131 , 1185..1338

Amore2p4_006966 90 1702 1 -175, 223..853, 901..1702

Amore2p4_006968 91 1342 1..109, 160..225, 277..290, 344-459, 508..1339

Amore2p4_006972 92 2454 1 .501 , 566-595, 685-1211 , 1272-2454

1..172, 224-385, 476-529, 576-627, 781..944, 1114-1254,

Amore2p4_006999 93 2224

1303..1338, 1392..2224

Amore2p4_007100 94 1735 1..137, 201..320, 378..517, 569..691 , 728..1090, 1155-1735

Amore2p4_007160 95 2715 1..1104, 1228-2715

Amore2p4_007161 96 1590 1..1590

Amore2p4_007162 97 1728 1..1728

Amore2p4_007233 98 2481 1..414, 475..812, 861.1912, 2312..2481

Amore2p4_007394 99 1774 1..326, 376..1547, 1629-1774

1..196, 246-272, 325-368, 426-699, 756-838, 894-1160,

Amore2p4_007580 100 1506

1228..1506

Amore2p4_007711 101 1867 1..261 , 309..426, 480.757, 807..979, 1030-1117, 1169-1867

Amore2p4_007744 102 1442 1..635, 746-1442

Amore2p4_007751 103 1471 1..215, 269-588, 662-949, 1005..1471

Amore2p4_007832 104 1122 1 -431 , 483-580, 638..1122

Amore2p4_007841 105 1635 1..1372, 1490-1635

1..123, 326..470, 526-575, 626..1005, 1059 .1454, 1507-1617,

Amore2p4_007951 106 2037

1713-2037

Amore2p4_007971 107 1629 1..253, 315-1456, 1507..1629

Amore2p4_008014 108 1293 1..253, 304..1124, 1180-1293

Amore2p4_008166 109 974 1..458, 507..690, 768-974

Amore2p4_008540 110 1379 1.77, 129..418, 463..490, 575..1379

Amore2p4_008644 111 1491 1..198, 250..1491

Amore2p4_008662 112 1794 1..100, 152..667, 751..1086, 1136-1794

Amore2p4_008748 113 3347 1..114, 208.790, 842..1105, 1279-1420, 1490..3347

Amore2p4_008830 114 1825 1..202, 253..420, 471..585, 636-862, 924-1825

Amore2p4_008851 115 1797 1..1797

1..101 , 159..209, 277-422, 481..562, 614..654, 739-912,

Amore2p4_008948 116 4329 967..2033, 2084..2237, 2312..2434, 2487-2538, 2599-2736,

2798..2816, 2872..3717, 3773-3856, 3919..3957, 4006..4329

Amore2p4_008958 117 1462 1..197, 256-368, 433-482, 531 .617, 672-856, 931 .1462

Amore2p4_008975 118 2777 1..141 , 200.710, 770-1840, 2623-2774

Amore2p4_008976 119 899 1 -59, 119-896

Amore2p4_009011 120 745 1 -261 , 338.745

Amore2p4_009098 121 1425 1..570, 629-823, 873-1299, 1352..1425

Amore2p4_009125 122 1314 1..67, 116-1314 Amore2p4_009129 123 1148 1..620, 677..1148

Amore2p4_009160 124 1168 1..41 , 165..410, 483..6Θ4, 735..1168

Amore2p4_009209 125 1666 1 .231 , 281. 1666

Amore2p4_009251 126 2063 1..255, 401..623, 688..1934, 1992..2063

Amore2p4_009309 127 2005 1..86, 193..668, 718..1100, 1151..2005

Amore2p4_009329 128 2689 1..298, 347..790, 861..956, 1004..2398, 2493..2689

Amore2p4_009350 129 1741 1..600, 695..1055, 1144..1610, 1670 .1741

1..4, 58..218, 273..324, 371..445, 501..637, 699.759, 807..905,

Amore2p4_009402 130 2209

955..1003, 1054 .1109, 1160..1687, 1734..2209

Amore2p4_009432 131 1404 1..65, 117 .389, 450..463, 599..1404

Amore2p4_009623 132 1497 1 .651 , 770..1160, 1253..1497

Amore2p4_009741 133 825 1..825

Amore2p4_009764 134 2158 1..217, 363..1652, 1721..2022, 2120 .2158

1 .188, 257.333, 38S..465, 515..564, 617.1000, 1065. 1222,

Amore2p4_009811 135 2353

1286 .1940, 2005..2180, 2305..2353

Amore2p4_009906 136 1152 1. 1152

Amore2p4_009952 137 2046 1 .132, 185..604, 661..998, 1050..2046

Amore2p4_009985 138 1681 1..159, 316 .501 , 553..9Θ8, 1238 .1548, 1605..1681

Amore2p4_010004 139 1169 1..533, 647. 1169

Amore2p4_010031 140 1429 1 .81 , 136..177, 226..340, 395..663, 725..1114, 1172..1429

Amore2p4_010080 141 2975 1..275, 476..2975

Amore2p4_001036 142 1835 1..953, 1004..1835

Amore2p4_001570 143 737 1..243, 318.737

Amore2p4_002213 144 740 1..42, 215..536, 601.740

Amore2p4_004667 145 629 1..211 , 268..62Θ

Amore2p4_006963 146 603 1..603

Amore2p4_009682 147 534 1..534

AMORE_00005 148 1551 1 .201 , 268. 1551

AMORE_00026 149 1429 1.708, 759..808, 865..1429

1 .112, 166..297, 385..437, 512..1369, 1420..1563, 1621..1971 ,

AMORE_00036 150 2556

2032..2201 , 2259..2319, 2383..255Θ

A ORE_00388 154 2067 1..153, 206..625, 682..1019, 1071 .2067

AMORE_00389 155 2046 1..132, 185..604, 661..998, 1050..2046

AMORE_00428 157 1170 1..285, 420..527, 574..1170

AMORE_00467 158 1122 1..431 , 483..580, 638. 1122

AMORE_2_00003 159 1660 1..201 , 291..995, 1460..1660

AMORE_2_00191 161 2086 1..201 , 291..995, 1629..1989, 2055..2086

1.78, 289..491 , 615..630, 1187.1462, 1516. 1541 , 1704..2086,

A ORE_2_00220 162 4497

2165..2332, 2475..3Θ32, 4022..4497

AMORE_3_00008 164 1133 1. 145, 201..250, 301..680, 734 .1133

AMORE_3_00009 165 1115 1..127, 183..232, 283..6Θ2, 716..1115

AMORE_3_00050 167 1055 1..138, 197.707, 767.1056

AMORE_3_00064 168 1394 1..146, 197.295, 356..1394

AMORE_3_00065 169 1391 1. 143, 194..292, 353..1391

AMORE_3_00077 170 1525 1..367, 419..844, 893..1053, 1103 .1525

AMORE_3_00113 172 1866 1 .1866

AMORE_3_00117 173 3347 1. 114, 208.790, 842..1074, 1127 .1229, 1279..1420, 1490..3347 AMORE_3_00147 174 1404 1..65, 120..389, 450..463, 599..1404

Amore2p4_000048 175 1866 1..505, 558.743, 808..851 , 904..1161 , 1225..1275, 1327..1866

Amore2p4_000900 176 778 1..497, 610.778

Amore2p4_002603 177 1683 1..1683

Amore2p4_003018 178 1434 1..1434

Amore2p4_004164 179 1428 1 .1332, 1387. 1428

Amore2p4_004649 180 1679 1..388, 441..1330, 1398 .1679

Amore2p4_004651 181 1917 .350, 411..942, 994..1917

Amore2p4_004752 182 1690 1..93, 157..607, 690..1690

1..51 , 110..195, 249..37Θ, 422..605, 798..Θ37, 1162..1623,

Amore2p4_004934 183 4319

1674..3556, 3618..4319

Amore2p4_005333 184 2382 1..2382

Amore2p4_006338 185 1333 1..241 , 295.770, 848..1333

Amore2p4_006377 186 1719 1..357, 410..1107, 1159..1370, 1451..1719

Amore2p4_006421 187 580 1..254, 337..580

1..119, 215..299, 354..519, 575..1785, 1960..2626, 2684..3452,

Amore2p4_006786 188 5676

3587..3Θ32, 4082..4361 , 4408..5117, 5179..5676

Amore2p4_007119 189 1889 1..343, 387..978, 1023 .1636, 1681..1889

1..291 , 357..458, 621..631 , 718..836, 88S..954, 1025..1201 ,

Amore2p4_007297 190 2467

1258..1268, 1327..1475, 1529 .1832, 1891..2196, 2316..2467

Amore2p4_007685 191 3187 1..1149, 1207 .1395, 1450..2086, 2136 .3187

Amore2p4_008937 192 1933 1. 1225, 1316..1589, 1648..1933

Amore2p4_009046 193 828 1..828

Amore2p4_009583 194 1740 1..234, 287..503, 565..Θ53, 1012 .1740

Table 2B. List of genes of Rhizomucor pus/7/us with reference to exon boundaries

Rhipu1p4_009465 675 1768 1..850, 920..1056, 1142..1768

Rhipu1p4_009591 676 2166 1..478, 538. 1363, 1428. 2166

Rhipu1 p4_009694 677 464 1..324, 384..4Θ4

1 .189, 25Θ..483, 551.713, 777..92Θ, 982..1248, 1310..1342,

Rhipu1 p4_009816 678 3545

1399..1482, 1542..1701 , 1761..2563, 2628..3254, 3337..3545

1..195, 257..481 , 561.723, 808..862, 932..1224, 1298..1330,

Rhipu1 p4_009817 679 3586 1383..1484, 1549..1705, 1778..2400, 2480..2599, 2670..3290,

3450..3586

1..366, 462..661 , 722..819, 88Θ..972, 1041..1127, 1196 .1277,

Rhipu1p4_009846 680 1364

1334..1364

Rhipu1p4_009848 681 729 1.729

Rhipu1p4_009849 682 729 1.729

Rhipu1p4_009855 683 1433 1..540, 610..862, 937..1023, 1091..1177, 1255..1333, 1400..1433

1..493, 594.752, 837..886, 948..1062, 1129..1227, 1294. 1446,

Rhipu1p4_009856 684 3149

1533..2270, 2332..3149

1..544, 607..659, 766..871 , 935..Θ84, 1060 .1273, 1331 .1477,

Rhipu1p4_009857 685 3176

1547..2224, 2302..3176

1 .173, 232..303, 394..574, 668.701 , 781 .1471 , 1545..1672,

Rhipu1 p4_009921 686 2084

1750..2084

Rhipu1p4_010133 687 1629 1. 141 , 213..440, 510..597, 659.733, 838 .852, 923..1629

Rhipu1 p4_010258 688 1701 1..465, 536..903, 971..1183, 1245..1701

Rhipu1 p4_010274 689 936 1..936

Rhipu1p4_010316 690 1448 1..402, 472..633, 688..950, 1019..1116, 1186..1448

Rhipu1 p4_010497 691 1636 1..630, 727..829, 900..1636

1..126, 200..295, 365.776, 843..916, 977. 1162, 1214..1257,

Rhipu1p4_010634 692 2729 1328..1656, 1722..1872, 1941..2075, 2137..2277, 2337..2495,

2589..266Θ, 2726..2729

1..125, 189..240, 332..398, 468..5Θ6, 639..887, 962..1067,

Rhipu1 p4_010639 693 2113

1131..1170, 1226..1503, 1569..1844, 1912..2113

Rhipu1 p4_010652 694 582 1..172, 245..344, 417..582

1..155, 258..391 , 453.-673, 745..879, 940..1194, 1257 .1338,

Rhipu1p4_010805 695 1586

1399..1586

Rhipu1 p4_010833 696 688 1..97, 162..688

RHIPU_1_00042 697 1011 1..615, 688. 1011

RHIPU_1_00044 698 1124 1..341 , 401..1124

RHIPU_1_00049 699 1159 1..265, 324..494, 575.701 , 769..876, 944..1016, 1076..1159

RHIPU_1_00065 700 1257 1..207, 381..554, 647..1048, 1108..1257

1..120, 192..297, 361..400, 452..5Θ6, 604.764, 834..1067,

RHIPU_1_00070 701 1696

1128..1398, 1458..1497, 1576..1619, 1682..1696

RHIPU_1_00076 702 1371 1..120, 194..383, 445..6Θ2, 727..819, 861..1155, 1223..1371

RHIPU_1_00081 703 1557 1..117, 188..323, 363..536, 607..815, 911..1420, 1507..1557

1.71 , 140..321 , 407. 511 , 588.713, 819..896, 967..1109,

RHIPU_1_00098 704 2972

1170..1550, 1613. 1648, 1704..2972

1..157, 220..486, 547..Θ35, 840..1094, 1157..1364, 1439..1574,

RHIPU_1_00113 705 2847

1644 .1702, 1773..2023, 2090..2239, 2401..2847

RHIPU_1_00123 706 2637 1..287, 339.443, 502..518, 586.706, 766..1617, 1725..2637

1..153, 257.478, 561.723, 808..827, 912. 1191 , 1298 .1330,

RHIPU_1_00190 707 3586 1383..1484, 1549 .1705, 1778..2395, 2463.-2599, 2670..3290,

3450..3586

RHIPU_1_00199 708 3246 1 -871 , 928..1070, 1127..1323, 1391..3134, 3199..3246

Table 2C. List of genes of Calcarisporiella thermophila with reference to exon boundaries

Genomic Genomic

Gene ID sequence sequence Exon boundaries (nucleotide positions) and exons

(SEQ ID NO:) length

Calth2p4_000015 1033 2518 1..216, 282..546, 618. 2518

Calth2p4_000019 1034 2435 1. 541 , 613..911 , 988. 1181 , 1257..1561 , 1624..2435

Calth2p4_000151 1035 1668 1..598, 658..953, 1039..1668

Calth2p4_000607 1036 1372 1 .1200, 1268..1372

1..76, 169..234, 284. 539, 646.722, 799..920, 991..1078,

Calth2p4_000644 1037 2818

1151..1709, 1797.1951 , 2046..2198, 2276.-2343, 2420..2818

Calth2p4_000661 1038 1380 1..681 , 751..1380

Calth2p4_000711 1039 1194 1..1101 , 1168..1194

Calth2p4_000722 1040 1405 1..260, 325.494, 579.722, 788..1194, 1259. 1405

Calth2p4_000723 1041 1412 1..260, 333..502, 572.715, 778..1184, 1251..1412

Calth2p4_000727 1042 1265 1..360, 424.746, 812..1265 Calth2p4_001059 1043 1055 1..206, 274..286, 351..535, 600..699, 760..842, 911..1055

1. 182, 248. 274, 351..548, 625.724, 782..8Θ4, 931.1062,

Calth2p4_001061 1044 1331

1250..1331

Calth2p4_001108 1045 1410 1..260, 326..495, 575.718, 789..1156, 1219..1410

Calth2p4_001243 1046 618 1..618

Calth2p4_001367 1047 2270 1..232, 372..514, 574..6Θ2, 730..1042, 1161..2270

Calth2p4_001374 1048 1086 1..298, 374.735, 803..907, 976..1086

Calth2p4_001438 1049 1263 1..357, 417.736, 789..1263

Calth2p4_001511 1050 1202 1..626, 707.1202

1..374, 438..500, 593..Θ94, 756..95S, 1023..1110, 1170..1249,

Calth2p4_001837 1051 1661

1310..1470, 1524..1661

Calth2p4_001932 1052 1656 1..293, 379..606, 673..1052, 1115..1656

Calth2p4_001948 1053 735 1..263, 325..481 , 538..648, 724.735

Calth2p4_002135 1054 1289 1. 281 , 334..890, 949..1289

Calth2p4_002336 1055 958 1..251 , 308..505, 566.745, 811..958

Calth2p4_002340 1056 1027 1..194, 280..477, 540..803, 886..1027

Calth2p4_002342 1057 1177 1..62, 130..411 , 505..885, 968..1052, 1118 .1177

Calth2p4_002369 1058 930 1..784, 848..930

Calth2p4_002419 1059 710 1..87, 179..395, 489..S95, 666.710

Calth2p4_002468 1060 907 1..375, 464..907

Calth2p4_002505 1061 1757 1..891 , 969..1757

Calth2p4_002595 1062 1428 1..1194, 1265 .1291 , 1363..1428

Calth2p4_002647 1063 354 1..354

1..194, 287..327, 395..474, 548..609, 675..810, 87Θ..998,

Calth2p4_002791 1064 3589 1064..1108, 1193..2719, 2793..3132, 3200..3327, 3397..3491 ,

3556. 3589

Calth2p4_002985 1065 690 1..690

Calth2p4_002987 1066 1274 1..395, 459..608, 676..88Θ, 954..1274

Calth2p4_003112 1067 1149 1..427, 502..818, 913..1149

Calth2p4_003433 1068 1087 1 .115, 182..269, 399..460, 528. 648, 709..801 , 868..1087

1..408, 486..519, 576..653, 724..1001 , 1061..1190, 1252..1361 ,

Calth2p4_003474 1069 2396

1421..1433, 1513..1789, 1846..2005, 2066..2179, 2262..2396

Calth2p4_003501 1070 1399 1..586, 661..854, 920..1399

Calth2p4_003508 1071 1105 1.750, 827..1105

Calth2p4_003526 1072 546 1..546

Calth2p4_003652 1073 958 1..824, 889..958

Calth2p4_003748 1074 1462 1..267, 376.771 , 845..901 , 990..1006, 1084..1462

1..347, 408..484, 562.765, 840..1134, 1203..1575, 1658..1897,

Calth2p4_003998 1075 2210

1956..2210

Calth2p4_004047 1076 708 1.371 , 429.708

Callh2p4_004227 1077 2234 1.761 , 837..1200, 1280..1490, 1585..2234

1.667, 761.844, 907..1127, 1214..1407, 1472..1944, 2015..2515,

Calth2p4_004392 1078 3333

2582..2Θ53, 2720..3255, 3325.-3333

Calth2p4_004418 1079 648 1.150, 215..466, 535. 648

1.128, 183..380, 462..603, 666..807, 883..903, 986..991 ,

Calth2p4_004472 1080 2192

1044 .1193, 1263 .1441 , 1512..1617, 1688..1921 , 1996..2192

Calth2p4_004506 1081 2307 1.262, 329..471 , 545 .633, 712..1030, 1109 .1289, 1364..2307

Calth2p4_004602 1082 1378 1.580, 661.971 , 1039..1152, 1229..1378 Calth2p4_004612 1083 2390 1 .371 , 461..537, 608..817, 883..1880, 1953..2390

Calth2p4_004793 1084 2783 1..187, 263.728, 794..1847, 1917..2783

Calth2p4_005043 1085 1999 1..260, 324..400, 474..1345, 1429..1671 , 1757..1999

Calth2p4_005058 1086 1361 1..159, 234..1361

Calth2p4_005092 1087 994 1..414, 488..994

Calth2p4_005516 1088 1513 1..983, 1052..1160, 1223..1513

Calth2p4_005549 1089 1701 1..299, 373.773, 852 .868, 963..1267, 1340..1360, 1452..1701

Calth2p4_005550 1090 1791 1..323, 391.791 , 865..881 , 964..1268, 1337..1357, 1467..1791

Calth2p4_005572 1091 825 1..825

Calth2p4_005651 1092 2996 1..403, 474..1309, 1382..1767, 1833..2076, 2139..2996

Calth2p4_005835 1093 1600 1..422, 497..Θ94, 783..835, 936..949, 1031..1600

Calth2p4_005946 1094 1008 1..1008

1..45, 126..232, 322 .411 , 480.707, 775..910, 982..1122,

Calth2p4_006010 1095 2096

1223..1483, 1553..1588, 1663..1743, 1815. 2096

Calth2p4_006172 1096 2339 1..976, 1083. 1576, 1643..1985, 2059..2339

Calth2p4_006230 1097 1334 1..463, 526. 845, 911..1027, 1090..1 137, 1212..1334

Calth2p4_006326 1098 1513 1..291 , 365..865, 926..1037, 1139..1344, 1424..1513

Calth2p4_006327 1099 1658 1..93, 163..197, 317..550, 628..1159, 1286. 1462, 1551..1658

Calth2p4_006505 1100 1185 1 .1185

1..105, 199..347, 433..465, 528..57Θ, 651..808, 882..1103,

Calth2p4_006699 1101 1740

1181..1442, 1563..1602, 1682..1740

Calth2p4_006734 1102 1821 1.75, 169..536, 614..948, 1015 .1364, 1450..1821

Calth2p4_006824 1103 1462 1..331 , 415..526, Θ27..644, 721..1462

Calth2p4_006971 1104 1589 1..460, 523..651 , 730..1019, 1111..1213, 1288..1589

Calth2p4_006976 1105 2905 1..914, 980..2675, 2750..2905

Calth2p4_007112 1106 1427 1..169, 235..471 , 528.739, 801 .1427

Calth2p4_007167 1107 564 1..564

Calth2p4_007200 1108 1795 1..509, 602.748, 823..1795

Calth2p4_007231 1109 601 1 .501 , 572..601

Calth2p4_007318 1110 1107 1..84, 211 .312, 37Θ..549, Θ35..989, 1061..1107

Calth2p4_007331 1111 1965 1..507, 587.775, 824..1526, 1628..1965

Calth2p4_007343 1112 1011 1..756, 847 .1011

1..342, 449..1861 , 1919 .2041 , 2120 .2167, 2286..2373,

Calth2p4_007472 1113 4055

2455..2758, 2816..2826, 3259..3578, 3663..4055

Calth2p4_007748 1114 1095 1..445, 494.741 , 812..922, 985..1095

Calth2p4_007756 1115 587 1..410, 491..587

1..307, 380..471 , 530..618, 677.791 , 851..1040, 1106..1409,

Calth2p4_007773 1116 3153

1486..1821 , 1887 .2130, 2193..2328, 2396..3153

Calth2p4_007802 1117 690 1..36, 127..690

Calth2p4_007854 1118 1244 1..632, 691..907, 975..1244

Calth2p4_007999 1119 1896 1..154, 221..640, 707..838, 899..1146, 1207. 1896

Calth2p4_008042 1120 354 1..354

Calth2p4_008043 1121 342 1..342

Calth2p4_008233 1122 505 1..253, 316..424, 493..505

1..269, 339..415, 476..679, 755..1043, 1109. 1481 , 1571 .1609,

Calth2p4_008588 1123 2207

1675..1881 , 971..2207

1. 196, 270..287, 352.528, 598..674, 742..967, 1025 .1222,

Calth2p4_008672 1124 1820

1285..1820 Calth2p4_008721 1125 1140 1. 1140

Calth2p4_008722 1126 1059 1..1059

1..205, 775..984, 1047 .1166, 1251..1297, 1362 .1635,

Calth2p4_008736 1127 2631

1694. 1747, 1815. 2063, 2150..2631

Calth2p4_008767 1128 1700 1..115, 192..506, 606..1456, 1521..1700

Calth2p4_009027 1129 1255 1..348, 408..730, 796..1255

Calth2p4_009092 1130 2346 1..756, 921. 1153, 1242 .1653, 1726..1911 , 2008..2346

Calth2p4_009254 1131 904 1..230, 292..904

Calth2p4_009259 1132 1437 1..412, 484..506, 573..839, 904..1437

Calth2p4_009317 1133 1100 1..468, 552..1100

Calth2p4_009862 1134 1221 1..1221

Calth2p4_010053 1135 1061 1..609, 686.796, 878 .892, 972..1061

Calth2p4_010222 1136 639 1..405, 490..639

Calth2p4_010236 1137 1686 1..1686

Calth2p4_010277 1138 2552 1 .115, 183..224, 287.393, 463..1012, 1099..2552

Calth2p4_010372 1139 1279 1..547, 619.703, 760..1279

Calth2p4_010526 1140 2414 1..461 , 550..626, 692..1893, 1956..2414

Calth2p4_010910 1141 868 1..116, 181..868

Calth2p4_010972 1142 529 1..105, 190. 351 , 431..529

Calth2p4_010979 1143 2068 1..381 , 454..846, 914..2068

Calth2p4_011171 1144 753 1.753

1..64, 123..280, 334..621 , 692..88Θ, 958..1221 , 1285..1412,

Calth2p4_011322 1145 13022 1486. 1685, 1744..5783, 5852..10426, 10493..12079,

12157.12237, 12308..12485, 12574..13022

Calth2p4_011430 1146 1214 1..592, 656..97S, 1056. 1214

Calth2p4_011483 1147 1040 1..121 , 219..275, 3S2..489, 574..651 , 721..897, 964..1040

Calth2p4_011624 1148 867 1..867

Calth2p4_005050 1149 1135 1 .141 , 208..309, 371..583, 657..1005, 1074..1135

1 .178, 258. 275, 345..400, 475 .595, 660.784, 855..1029,

Calth2p4_008288 1150 1833

1103 .1833

CALTH_1_05687 1151 2858 1..265, 336..1171 , 1244..1629, 1695..1938, 2001..2858

CALTHJJ 0213 1152 2210 1..257, 34Θ..422, 488..1689, 1752..2210

CALTH_2_00006 1153 2858 1..265, 336..1171 , 1244 .1629, 1695. 1875, 2001..2858

1..278, 385..461 , 538..Θ59, 730..817, 890..1448, 1536. 1690,

CALTH_2_00035 1154 2557

1785. 1937, 2015..2082, 2159..2557

CALTH_2_00045 1155 849 1..372, 457..849

CALTH_2_00054 1156 1659 1..374, 452.786, 853..1202, 1288..1659

CALTH_2_00167 1158 2068 1..381 , 454..552, 613..846, 914..2068

1..271 , 334..453, 538..584, 649 .922, 981. 1034, 1102..1350,

CALTH_2_00178 1159 1918

1437.1918

CALTH_2_00249 1160 1262 1..383, 447..S96, 6Θ4..877, 942..1262

1..115, 182..269, 399..460, 528..648, 709..801 , 868..1036,

CALTH_2_00251 1161 1537

1418..1537

CALTH_2_00259 1162 1027 1..194, 280..477, 540..565, 650..803, 886..1027

Calth2p4_000004 1163 1076 1. 211 , 304 .673, 749..1076

Calth2p4_000258 1164 1763 1..154, 221 .448, 514..1169, 1233..1763

Calth2p4_000725 1165 897 1..897

Calth2p4_000916 1166 1726 1..256, 324..1726 Calth2p4_001437 1167 1905 1..615, 684..1169, 1255..1574, 1623..1905

Calth2p4_001751 1168 1710 1..148, 215..343, 404..1710

1..46, 114 .357, 418..639, 701..822, 897..1455, 1517..1791 ,

Calth2p4_001908 1169 3185

1860..2458, 2526..3185

Calth2p4_001924 1170 1199 1..107, 172..833, 904. 1199

1..73, 128. 195, 272..477, 570..1654, 1712..1973, 2046..2433,

Calth2p4_002556 1171 3254

2511. 2982, 3064 .3118, 3203..3254

Calth2p4_002583 1172 1742 1 .1001 , 1055..1742

Calth2p4_002645 1173 2085 1 .183, 427..633, 697..990, 1809..1918, 1962..2085

Calth2p4_003109 1174 575 1..446, 560..575

Calth2p4_003138 1175 1940 1. 621 , 699..1940

Calth2p4_003764 1176 1731 1..1329, 1429..1731

Calth2p4_004510 1177 1844 1..97, 193..252, 340..567, 646..1844

Calth2p4_005465 1178 1830 1..1830

Calth2p4_006950 1179 1357 1..256, 318..1106, 1212..1357

Calth2p4_007279 1180 1731 1 .1731

Calth2p4_007579 1181 732 1..475, 536.732

Calth2p4_007709 1182 1724 1..97, 178..237, 301..1724

Calth2p4_007829 1183 1226 1..154, 219 .951 , 1145..1226

Calth2p4_008551 1184 1223 1..192, 260..361 , 423..638, 724..1078, 1144..1223

Calth2p4_008658 1185 1008 1 -150, 206..307, 348..581 , 652..1008

Calth2p4_008991 1186 1839 1..1839

Calth2p4_009788 1187 1393 1..196, 273..651 , 717.807, 867.1204, 1294..1393

Calth2p4_010203 1188 604 1..104, 175..210, 271..604

Calth2p4_010278 1189 789 1.789

Calth2p4_010337 1190 1177 1..195, 255-356, 423-629, 691 -1039, 1113..1177

Calth2p4_010659 1191 1854 1..1854

Calth2p4_010906 1192 1722 1..1722

Calth2p4_010999 1193 1674 1..1674

Calth2p4_011211 1194 1815 1..473, 521..1310, 1403-1697, 1802..1815

Calth2p4_011294 1195 1842 1..1842

Calth2p4_011333 1196 1839 1..1839

Calth2p4_011384 1197 1679 1..592, 691 -986, 1056-1679

Calth2p4_011450 1198 1731 1..1731

Calth2p4_011519 1199 1719 1..1719

Calth2p4_011520 1200 695 1..67, 124..695

Calth2p4_011542 1201 1733 1..160, 232-1212, 1288- 1733

Calth2p4_011637 1202 1728 1..1728

[00228] The present invention is illustrated in further details by the following non-limiting examples.

Example 1: Fermentation of the organism [00229] In general, for each species, starter mycelium was grown in rich medium (either mycological broth or yeast malt broth (the latter being indicated with YM)) and then washed with water. The starter was then used to inoculate different liquid media or solid substrate and the resulting mycelium was used for RNA extraction and library construction.

[00230] Following are the medium recipes and the solid substrates with a referenced source (if available) as well as a table (Table 3) listing the media variations, since in some cases the basic recipes of the referenced source have been altered depending on the species grown. This is then followed by a summary of the specific species as grown in the examples.

A. Mycological broth

Per liter: 10 g soytone, 40 g D-glucose, 1 mL Trace Element solution, Double-distilled water;

Adjust pH to 5.0 with hydrochloric acid (HCI) and bring volume to 1 L with double-distilled water.

Trace Element Solution contains 2 mM Iron(ll) sulphate heptahydrate (FeSO h^O), 1 mM Copper (II) sulphate pentahydrate (CuSCyShkO), 5 mM Zinc sulphate heptahydrate (ZnSC hbO), 10 mM Manganese sulphate monohydrate (MnSCy^O), 5 mM Cobalt(ll) chloride hexahydrate (CoCb'BFbO), 0.5 mM Ammonium molybdate tetrahydrate ((NH^MorC^I-bO), and 95 mM Hydrochloric acid (HCI) dissolved in double-distilled water.

B. Yeast-Malt broth (YM)

(Reference: ATCC medium No. 200)

Per liter: 3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g D-glucose, Double-distilled water to 1 L.

C. Trametes Defined Medium (TDM)

(Reference: Reid and Piace, "Effect of Residual lignin type and amount on biological bleaching of kraft pulp by Trametes versicolor". Applied Environmental Microbiology 60: 1395-1400, 1994.)

Per liter: 10 g D-glucose, 0.75 g L-Asparagine monohydrate, 0.68 g Potassium phosphate monobasic (KH2PO4), 0.25 g Magnesium sulphate heptahydrate (MgS04 ^# 7H ₂ 0), 15 mg Calcium chloride dihydrate (CaCl2'2H ₂ 0), 100 g Thiamine hydrochloride, 1 ml Trace Element solution, 0.5 g Tween™ 80, Double distilled water;

Adjust pH to 5.5 with 3 M potassium hydroxide and bring volume to 1 L with double-distilled water.

Table 3. Variations of TDM media used for library construction

Variation Description

TDM-1 Medium was prepared as in basic recipe described above.

TDM-2 Quantity of asparagine monohydrate was reduced to 0.15 g.

TDM-3 Manganese sulphate monohydrate was omitted from the medium.

The quantity of manganese sulphate monohydrate was raised to 0.2 mM final concentration in the

TDM-4

medium.

TDM-5 The quantity of copper (II) sulphate pentahydrate was raised to 20 μΜ.

TDM-6 Glucose was replaced with 10 g per liter of cellulose (Solka-Floc, 200FCC) TDM-7 Glucose was replaced with 10 g per liter of xylan from birchwood (Sigma Cat. # X-0502)

TDM-8 Glucose was replaced with 10 g per liter of wheat bran ¹ .

TDM-9 Glucose was replaced with 10 g per liter of citrus pectin (Sigma Cat. # P-9135).

TD -10 Tween™ 80 was omitted from the medium.

The double-distilled water was replaced with Whitewater ² collected from peroxide bleaching (which

TDM-11

occurs during the manufacture of fine paper).

TDM-12 The double-distilled water was replaced with Whitewater ² collected from newsprint manufacture.

TDM-13 Glucose was replaced with 5 g per liter of ground hardwood kraft pulp ³ .

TDM-14 The medium's pH was raised to 7.5.

TDM-15 The strain was incubated at 5°C above its optimum growth temperature.

TD -16 The strain was incubated at 10°C below its optimum growth temperature.

One half of the double-distilled water was replaced with Whitewater from newsprint manufacture.

TDM-17

Glucose was omitted.

Potassium phosphate monobasic was replaced with 5 mM phytic acid from rice (Sigma Cat. #

TDM-18

P3168).

TDM-19 Asparagine monohydrate was increased to 4 g per liter.

Asparagine monohydrate was increased to 4g per liter and glucose was replaced with 2% fructose.

TD -20

Asparagine monohydrate was increased to 4 g per liter; 100 mL of double-distilled water was

TDM-21

replaced with 100 mL kerosene ⁴ . Glucose was omitted.

Asparagine monohydrate was increased to 4 g per liter; 100 mL of double-distilled water was

TDM-22

replaced with 100 mL hexadecane (Sigma cat. # H0255). Glucose was omitted.

Asparagine monohydrate was increased to 4 g per liter; one half of the double-distilled water was

TDM-23 replaced with 25% Whitewater from newsprint manufacture plus 25% white water from peroxide bleaching. Glucose was omitted.

Asparagine monohydrate was increased to 4 g per liter and the quantity of manganese sulphate

TDM-24

monohydrate was raised to 0.2 mM final concentration in the medium.

Asparagine monohydrate was increased to 4 g per liter and manganese sulphate monohydrate was

TD -25

omitted from the medium.

TDM-26 Asparagine monohydrate was increased to 4 g per liter; and potassium phosphate monobasic was replaced with 5mM phytic acid from rice (Sigma Cat. # P3168).

TDM-27 Glucose was replaced with 10g per liter of olive oil (Sigma cat. # 01514)

One half of the double-distilled water was replaced with Whitewater from peroxide bleaching.

TDM-28

Glucose was omitted.

TDM-29 Glucose was replaced with 10 g per liter of tallow.

TDM-30 Glucose was replaced with 10 g per liter of yellow grease.

TDM-31 Glucose was replaced with 10 g per liter of defined lipid (Sigma cat. # L0288).

TD -32 Glucose was replaced with 50 g per liter of D-xylose.

TD -33 Glucose was replaced with 20 g per liter of glycerol and 20ml per liter of ethanol. TDM-34 Glucose was reduced to 1 g per liter and 10 g per liter of bran was added.

TDM-35 Glucose was reduced to 1 g per liter and 10 g per liter of pectin (Sigma Cat. # P-9135) was added.

TDM-36 Glucose was replaced with 10 g per liter of biodiesel.

TDM-37 Glucose was replaced with 10 g per liter of soy feedstock.

TDM-38 Glucose was replaced with 10g per liter of locust bean gum (Sigma cat # G0753).

One half of double-distilled water was replaced with a 1 :1 ratio of Whitewater from newsprint

TD -39

manufacture and white water from peroxide bleaching. Glucose was omitted.

TDM-40 The medium's pH was raised to 8.5.

One half of double-distilled water was replaced with Whitewater from peroxide bleaching; plus yeast

TDM-41

extract was added to 1 g per liter. Glucose was omitted.

TD -42 Glucose was replaced with 5 g per liter of yellow grease and 5 g per liter of soy feedstock

TDM-43 Glucose was replaced with 20g per liter of fructose.

Glucose was replaced with 10 g per liter of cellulose (Solka-Floc, 200FCC) plus 1 g per liter of

TDM-44

sophorose.

TDM-45 The medium's pH was raised to 8.84.

¹ Food grade wheat bran sourced from the supermarket was used.

² All Whitewaters were sourced from Quebec paper mills by PAPRICAN on the Applicant's behalf.

³ Hardwood kraft pulp was sourced from Quebec paper mills by PAPRICAN on the Applicant's behalf.

⁴ Kerosene was sourced from a general hardware store.

D. Asparagine Salts Medium (AS):

(Reference: Ikeda et al., "Laccase and Melanization in Clinically Important Cryptococcus Species Other Than

Cryptococcus neoformans", Journal of Clinical Microbiology 40: 1214-1218, 2002)

Per liter: 3.0 g D-glucose, 1.0 g L-Asparagine monohydrate, 3.0 g KH2PO4, 0.5 g Mg S04-7H20, 1 mg Thiamine.

Table 4: Variations of AS media used for library construction

E. Solid substrates used:

SS-1 5 g Wheat Bran.

SS-2 5 g Wheat bran plus 5 mL defined lipid.

SS-3 5 g Oat bran (food grade, sourced from supermarket).

[00231] The Chaetomium thermophilum strain was grown according to the methods described above under the following growth conditions: TDM-1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -13, -14, -15, -39; YM, whereby the following optimal growth temperature was used: 25°C.

[00232] The strains carrying the recombinant genes were grown according to the methods described above under the following growth conditions: minimal medium as described in Kafer et al., (1977, Adv. Genet. 19:33- 131) except that the salt concentrations were raised ten-fold and the glucose concentration was 150 grams per liter, at 30°C.

Example 2: Genome sequencing and assembly

[00233] Genomic DNA was isolated from mycelium when the growth culture had reached the mid log phase. Genomic DNA was sequenced using the Roche 454 Titanium technology (http://www.454.com) to a genome coverage of over 20-fold according to the instructions of the manufacturer. The sequences were assembled using the Newbler and Celera assemblers (http://sourceforge.net/apps/mediawiki/wgs-assembler).

Example 3: Building the cDNA libraries

[00234] Total RNA was isolated from fungal cells or mycelia when the growth cultures had reached the late log phase. The mycelia were collected by filtration through Miracloth and washed with water by filtration. The mycelia were padded dry using paper towels, and frozen in liquid nitrogen and stored at -80°C. To extract total RNA, the frozen mycelia or cells were ground to a fine powder in liquid nitrogen using pestle and mortar. Approximately 1-1.5 gram of frozen fungal powder was dissolved in 10 mL of TRIzol® reagent and RNA was extracted according to the manufacturer's protocol (Invitrogen Life Sciences, Cat. #15596-018). Following extraction, the RNA was dissolved at 1-1.5 mg/ml of DEPC-treated water.

[00235] The PolyATtract® mRNA Isolation Systems (Promega, Cat. #Z5300) was used to isolate poly(A)+RNA. In general, equal amounts of total RNA extracted from up to ten culture conditions were pooled. One milligram of total RNA was used for isolation of poly(A)+RNA according to the protocol provided by the manufacturer. The purified poly(A)+RNA was dissolved at 200-500 pg/mL of DEPC-treated water.

[00236] Five micrograms of poly(A)+RNA were used for the construction of cDNA library. Double-stranded cDNA was synthesized using the ZAP-cDNA® Synthesis Kit (Stratagene, Cat. #200400) according to the manufacturer's protocol with the following modifications. An anchored oligo(dT) linker-primer was used in the first-strand synthesis reaction to force the primer to anneal to the beginning of the poly(A) tail of the mRNA. The anchored oligo(dT) linker-primer has the sequence:

5' -GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTTVN-3' where V is A, C, or G and N is A, C, G, or T. A second modification was made by adding trehalose at a final concentration of 0.6 M and betaine at a final concentration of 2 M in the buffer of the first-strand synthesis reaction to promote full-length synthesis. Following synthesis and size fractionation, fractions of double-stranded cDNA with sizes longer than 600 bp were pooled. The pooled cDNA was cloned directionally into the plasmid vector BlueScript KS+® (Stratagene) or a modified BlueScript KS+ vector that contained Gateway® (Invitrogen) recombination sites. The cDNA library was transformed into E. coli strain XL10-Gold ultracompetent cells (Stratagene, Cat. #Z00315) for propagation.

[00237] Bacterial cells carrying cDNA clones were grown on LB agar containing the antibiotic ampicillin for selection of plasmid-borne bacteria and X-gal and IPTG to use the blue/white system to screen for the presence cDNA inserts. The white bacterial colonies, those carrying cDNA inserts, were transferred by a colony-picking robot to 384-well MTP for replication and storage. Clones that were to be analyzed by sequencing were transferred to 96-well deep blocks using liquid-handling robots. The bacteria were cultured at 37°C with shaking at 150 rpm. After 24 hours of growth, plasmid DNA from the cDNA clones was prepared by alkaline lysis and sequenced from the 5' end using ABI 3730x1 DNA analyzers (Applied Biosystems). The chromatograms obtained following single-pass sequencing of the cDNA clones were processed using Phred (available at http://www.phrap.org) to assign sequence quality values, Lucy as described in Chou and Holmes (2001 , Bioinformatics, 17(12) 1093-1104) to remove vector and low quality sequences, and Phrap (available at http://www.phrap.org/) to assemble overlapping sequences derived from the same gene into contigs.

Example 4: Annotations

[00238] An in-house automated annotation pipeline was used to predict genes in the assembled genome sequence. The analysis pipeline used in part the ab initio tool Genemark® (http://exon.biology.gatech.edu/) for prediction. It also used the predictor Augustus (http://augustus.gobics.de/) trained on de novo assembled sequences and orthologous sequences for gene finding. Sequence similarity searches against the mycoCLAP® (http://cubique.fungalgenomics.ca/mycoCLAP/) and NCBI non-redundant databases were performed with BLASTX as described in Altschul et al., (1997) (Nucleic Acids Res. 25(17): 3389-3402). Proteins encoding biomass-degrading enzymes possess conserved domains. We used the domains available at the European Bioinformatics Institute (www.ebi.ac.uk/Tools/lnterProScan/) to assist in the identification of target enzymes.

[00239] Proteins targeted to the extracellular space by the classical secretory pathway possess an N- terminal signal peptide, composed of a central hydrophobic core surrounded by N- and C- terminal hydrophilic regions. We used Phobius (available at http://phobius.cgb.ki.se) and SignalP® version 3 (available at http://www.cbs.dtu.dk/services/SignalP) to recognize the presence of signal peptides encoded by the cDNA clones. The tools TargetP® (available at http://www.cbs.dtu.dk/services/TargetP) and Big-PI Fungal Predictor (available at http://mendel.imp.ac.at/gpi/fungi_server.html) were used to remove sequences that encode proteins which are targeted to the mitochondria or bound to the cell wall. Finally, sequences predicted to encode soluble secreted proteins by these automated tools were analyzed manually. Clones that comprise full-length cDNAs which are predicted to encode soluble secreted proteins were sequenced completely. For genes identified from the genome sequence, oligonucleotide primers specific to the target genes were designed and used to PCR amplified the target genes from double-stranded cDNA or genomic DNA. The PCR amplified products were cloned into an appropriate expression vector for protein production in host cells. The genomic, coding and polypeptide sequences were assigned SEQ ID NOs, annotations, general functions, protein activities, CAZy family classifications, as summarized in Tables 1A-1C. Where appropriate, carbohydrate-binding modules (CBMs) of particular interest for the degradation of biomass were also listed in Tables 1A-1C.

Example 5: Assays for characterization of polypeptides

[00240] Polypeptides of the present invention may be additionally cloned into an expression vector, expressed and characterized (e.g., in sugar release assays) for activity relating to their ability to breakdown and/or process biomass as described in WO/2012/92676, WO/2012/130950, WO/2012/130964, and WO/2013/181760 using appropriate substrates (e.g., acid pre-treated corn stover, hot water treated washed wheat straw, or hot water treated washed corn fiber substrate). Soluble sugars that are released can be analyzed for example by proton NMR.

[00241] A number of assays may be used to characterize the polypeptides of the present invention. Selected non-limiting examples of such assays are described and/or referenced below. Of course, other assays not explicitly mentioned or referenced here may also be used, and the expression "can be" used below is intended to reflect this possibility. Furthermore, a person of skill in the art would be able to modify or adapt these and other assays, as necessary, to characterize a particular polypeptide.

4-O-methyl-glucuronoyl methylesterase. Polypeptides of the present invention having this activity can be characterized for example as described in Li et al, FEBS Lett. (2007), 581 (21): 4029-35; Spanikova and Biely, FEBS Lett. (2006), 580(19): 4597-601.

11-beta-hydroxysteroid dehydrogenase 1 B. Polypeptides of the present invention having this activity can be characterized for example as described in Blum et al., Biochemistry (2003), 42(14):4108-17.

2-(R)-hydroxypropyl-CoM dehydrogenase (2-(2-(R)-hydroxypropylthio)ethanesulfonate dehydrogenase).

Polypeptides of the present invention having this activity can be characterized for example as described in Sliwa et al„ 6/oc/?em/sfry(2010), 49(16):3487-98.

Acetylxylan esterase. Polypeptides of the present invention having this activity can be characterized as described in Water et al., Appl Environ Microbiol. (2012), 78(10): 3759-62; Yang et al., International Journal of Molecular Sciences (2010), 11 (12): 5143-5151 ; or in US patent No. 8,129,590.

Adhesin protein Madl Polypeptides of the present invention having this activity can be characterized for example as described in Wang and St Leger, Eukaryot. Cell (2007), 6(5): 808-816.

Adhesin. Polypeptides of the present invention having this activity (reviewed in Dranginis et al., Microbiology and Molecular Biology Reviews (2007), 71 (2): 282-294) can be characterized using techniques well known in the art (e.g. adhesion assays).

Alcohol dehydrogenase [acceptor]. Polypeptides of the present invention having this activity can be characterized for example as described in Krog et al., PLoS One (2013) 8(3):e59188.

Aldonolactonase. Polypeptides of the present invention having this activity can be characterized for example as described in Beeson et al., Appl Environ Microbiol. (2011), 77(2): 650-6; Ishikawa et al„ J Biol Chem. (2008), 283(45): 31133-41.

Aldose 1-epimerase (mutarotase, aldose mutarotase). Polypeptides of the present invention having this activity can be characterized for example as described in Timson and Reece, FEBS Letters (2003), 543(1 -3):21 -24; and Villalobo et al., Exp. Parasitol. (2005) 110(3): 298-302.

Alkaline protease 2. Polypeptides of the present invention having this activity can be characterizedfor example as described in Gomaa, Braz J Microbiol. (2013) 44(2):529-37; or Yao et al., J Food Sci Technol. (2012), 49(5):626-31.

Allergen Asp f 15. Polypeptides of the present invention having this activity can be characterizedfor example as described in Bowyer et al., Medical Mycology (2007), 45(1): 17-26. Alpha-arabinofuranosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Poutanen et al. {AppI. Microbiol. Biotechnol. 1988, 28, 425-432) using 5 mM p- nitrophenyl alpha-L-arabinofuranoside as substrates. The reactions may be carried out in 50 mM citrate buffer at pH 6.0, 40°C with a total reaction time of 30 min. The reaction is stopped by adding 0.5 ml of 1 M sodium carbonate and the liberated p-nitrophenol is measured at 405 nm. Activity is expressed in U/ml. Furthermore, arabionofuranosidases may also be useful in animal feed compositions to increase digestibility. Corn arabinoxylan is heavily di-substituted with arabinose. In order to facilitate the xylan degradation it is advantageous to remove as many as possible of the arabinose substituents. The in vitro degradation of arabinoxylans in a corn based diet supplemented with a polypeptide of the present invention having alpha- arabinofuranosidase activity and a commercial xylanase is studied in an in vitro digestion system, as described in WO/2006/114094.

Alpha-fucosidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,637,490; in Zielke et al, J. Lab. Clin. Med. (1972), 79:164; or using commercially available kits (e.g., Alpha-L-Fucosidase (AFU) Assay Kit, Cat. No. BQ082A-EALD, BioSupplyUK).

Alpha-galactosidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2010/0273235 A1. Briefly, a synthetic substrate, 4-Nitrophenyl-a-D-galactoside is used and the release of p-Nitro-phenol is followed at a wavelength of 405 nm in a reaction buffer containing 100 mM sodium phosphate, 50 mM sodium chloride, pH 6.8 at 26°C.

Alpha-glucuronidase GH67. Polypeptides of the present invention having this activity can be characterized for example as described in Lee et al, J Ind Microbiol Biotechnol. (2012), 39(8): 1245-51 , or Nagy et al, J. Bacterid. (2002), 184: 4925-4929.

Alpha-rhamnosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Fujimoto et al, J Biol Chem. (2013), 288(17):12376-85; Rodriguez et al, J AppI Microbiol. (2010), 109(6): 2206-13; Grandits et al, J Mol Catal B Enzym. (2013), 92(100): 34^3.

Aminopeptidase Y. Polypeptides of the present invention having this activity can be characterized for example as described in Yasuhara et al, J. Biol. Chem. (1994) 269(18) : 13644-50.

Arabinan endo-1,5-alpha-L-arabinosidase A. Polypeptides of the present invention having this activity can be characterized for example as described in Flipphi et al, AppI. Microbiol. Biotechnol. (1993), 40: 318-326; and Leal and de Sa-Nogueira, FEMS Microbiol. Lett. (2004), 241 : 41 -48.

Arabinogalactanase. Polypeptides of the present invention having this activity can be characterized for example as described in Yamamoto and Emi, Methods in Enzymology (1988), 160: 719-725.

Arabinoxylan arabinofuranohydrolase (AXH) GH43. Polypeptides of the present invention having this activity can be characterized for example as described in Yoshida et al. Journal of Bacteriology (2010), 192(20): 5424- 5436.

Arabinoxylan arabinofuranosidase GH62. Polypeptides of the present invention having this activity can be characterized for example as described in Sakamoto et al. Applied Microbiology and Biotechnology (2011), 90(1): 137-146.

Aspartic protease. Polypeptides of the present invention having this activity can be characterized for example as described in Tacco et al, Med. Mycol. (2009), 47(8): 845-854; or in Hu et al. Journal of Bio edicine and Biotechnology (2012), 2012:728975.

Aspartic-type endopeptidase. Polypeptides of the present invention having this activity can be characterized for example as described in Tjalsma et al, J. Biol. Chem. (1999), 274: 28191-28197.

Aspergillopepsin-2. Polypeptides of the present invention having this activity can be characterized for example as described in Huang et al. Journal of Biological Chemistry (2000), 275(34): 26607-14. Avenacinase. Polypeptides of the present invention having this activity can be characterized for example as described in Kwak et al., Phytopathology (2010), 100(5): 404-14; or in Bowyer et al., Science (1995), 267(5196): 371-4.

Beta-galactosidase. Polypeptides of the present invention having this activity can be characterized for example using commercially available kits (e.g., β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer, Cat. No. E2000, Promega).

Beta-glucanase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication number US 2012/0023626 A1 ; or in US patent No. 8,309,338.

Beta-glucosidase. Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO/2007/019442; or by using a commercially available kit (e.g., Beta-Glucosidase Assay Kit, Cat. No. KA1611 , Abnova Corp).

Beta-glucuronidase. Polypeptides of the present invention having this activity can be characterized for example as described in Eudes et al., Plant Cell Physiology (2008), 49(9): 1331-41 ;or Michikawa et al., Journal of Biological Chemistry (2012), 287: 14069-14077.

Beta-hexosaminidase. Polypeptides of the present invention having this activity can be characterized for example as described in Wendeler and Sandhoff, Glycoconj J. (2009), (8):945-52.

Beta-mannanase. Polypeptides of the present invention having this activity can be characterized for example as described in European patent application No. EP 2261359 A1 ; or in PCT application publication No. WO2008009673A2.

Beta-mannosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Park et al., N. Biotechnol. (2011), 28(6): 639-48; Duffaud et al, Appl Environ Microbiol. (1997), 63(1): 169-77; or in Fliedrova et al. Protein Expr Purif. (2012), 85(2): 159-64.

Beta-xylosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Wagschal et al. Applied and Environmental Microbiology (2005), 71 (9): 5318-5323; or Shao et al, Appl Environ Microbiol. (2011), 77(3): 719-726.

Bifunctional alpha-arabinofuranosidase/beta-xylosidase GH43. Polypeptides of the present invention having this activity can be characterized for example as described in Viborg et al, AMB Express. (2013), 3(1):56; Shi et al, Biotechnol Biofueis (2013), 6(1):27; or Kim and Yoon, J Microbiol Biotechnol. (2010), (12): 1711-6.

Bifunctional xylanase/deacetylase. Polypeptides of the present invention having this activity can be characterized for example as described in Cepeljnik et al. Folia Microbiol. (2006), 51 (4): 263-267; US patent application publication No. US 2012/0028306 A1 ; US patent No. 7,759,102; or PCT application publication No. WO 2006/078256 A2; or Grozinger and Schreiber, Chem Biol. (2002), 9(1): 3-16.

Carbohydrate-binding cytochrome. Polypeptides of the present invention having this activity can be characterized for example as described in Yoshida et al, Appl Environ Microbiol. (2005) 71 (8): 4548-4555.

Carboxylesterase. Polypeptides of the present invention having this activity can be characterized for example using a commercially available kit such as the Carboxylesterase 1 (CES1) Specific Activity Assay Kit (ab109717) (Abeam, Cambridge, MA, USA).

Carboxypeptidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2007/0160711 A1 ; or in PCT application publication No. WO 1998/014599A1.

Cellobiohydrolase GH6. Polypeptides of the present invention having this activity can be characterized for example as described in Takahashi et al. Applied and Environmental Microbiology (2010), 76(19): 6583-6590. Cellobiohydrolase GH7. Polypeptides of the present invention having this activity can be characterized for example as described in Segato et al., Biotechnology for Biofuels (2012), 5:21 ; or Baumann et al., Biotechnol. for Biofuels (2011 ), 4:45; or Naran et al., Plant J.(2007), 50(1):95-107.

Cellobiose dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Schou et al., Biochem. J. (1998), 330: 565-571 ; or Baminger et al., J. Microbiol Methods. (1999), 35(3): 253-9.

Chitin deacetylase. Polypeptides of the present invention having this activity can be characterized for example as described in European patent application No. EP 0610320 B1.

Chitinase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 7,087,810.

Chitinase-3-like protein 1. Polypeptides of the present invention having this activity can be characterized for example as described in Dela Cruz et al., Cell Host Microbe (2012), 12(1):34-46.

Chitooligosaccharide deacetylase. Polypeptides of the present invention having this activity can be characterized for example as described in John et al., Proc Natl Acad Sci USA (1993), 90(2): 625-9.

Chitotriosidase-1. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 6,057,142.

Choline dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Gadda and McAllister-Wilkins, Appl. Environ. Microbiol. (2003) 69(4): 2126-32; or Takabe et al., J. Biol. Chem. (2003), 278 (7): 4932-42.

Cholinesterase. Polypeptides of the present invention having this activity can be characterized for example as described in Abass Askar et al., Canadian Journal Veterinary Research (2011), 75(4): 261-270; or Catia et al., PLoS One (2012), 7(3): e33975.

Cutinase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0028318 A1 ; or in Chen et al., J. Biol Chem. (2008), 283(38): 25854-62.

Cytochrome P450. Polypeptides of the present invention having this activity can be characterized for example as using commercially available kits (e.g., P450-GIO™ Assays, Promega); or as described in Walsky and Obach, Drug Metab Dispos. (2004), 32(6): 647-60.

Dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Mayer and Arnold, J. Biomol. Screen. (2002), 7(2): 135-140.

Dipeptidyl peptidase. Polypeptides of the present invention having this activity can be characterized for example as described in Ohara-Nemoto et al., J Biol Chem. (2014) Jan 7. [Epub ahead of print] PMID:

24398682.

Endo-1 ,3(4)-beta-glucanase (laminarinase). Polypeptides of the present invention having this activity can be characterized for example as described in Akiyama et al., J Plant Physiol. (2009), 166(16): 1814-25; or Hua et al., Biosci Biotechnol Biochem. (2011), 75(9): 1807-12.

Endo-1,4-beta-xylanase. Polypeptides of the present invention having this activity can be characterized for example as described in Song et al., Enzyme and Microbial Technology (2013). 52(3): 170-176. Endo-1,5-alpha-arabinanase Polypeptides of the present invention having this activity can be characterized for example as described in US patent publication No. US 2012/0270263. More particularly, this assay of arabinase activity is based on colorimetrically determination by measuring the resulting increase in reducing groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity can be calculated from the relationship between the concentration of reducing groups, as arabinose equivalents, and absorbance at 540 nm. The assay is generally carried out at pH 3.5, but it can be performed at different pH values for the additional characterization and specification of enzymes. Polypeptides of the present invention having this activity can also be characterized for example as described in Hong et al., Biotechnol Lett. (2009), 31 (9): 1439-43.

Endo-1 ,6-beta-glucanase. Polypeptides of the present invention having this activity can be characterized for example as described in Bryant et al., Fungal Genet Biol. (2007), 44(8): 808-17; or in Oyama et al., Biosci Biotechnol Biochem. (2006), 70(7): 1773-5.

Endo-beta-1 ,3-galactanase. Polypeptides of the present invention having this activity can be characterized for example as described in Kotake et al., J Biol Chem. (2011), 286(31): 27848-54. Ichinose et al., AppI Environ Microbiol. (2006), 72(5): 3515-3523.

Endo-beta-1 ,4-glucanase celB. Polypeptides of the present invention having this activity can be characterized for example as described in Baird et al., J Bacteriol. (1990), 172(3): 1576-1586; Jorgensen and Hansen, Gene. (1990), 93(1):55-60; Jagtap et al., "Characterization of a novel endo- -1 ,4-glucanase from Armillaria gemina and its application in biomass hydrolysis", AppI Microbiol Biotechnol. (2013).

Endochitinase Polypeptides of the present invention having this activity can be characterized for example as described in Wen et al., Biotechnol. Applied Biochem. (2002), 35: 213-219.

Endoglucanase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 8,063,267; Couturier et al., Microb Cell Fact. (2011 ), 10:103; Badieyan et al., Biotechnol Bioeng. (2012), 109(1): 31-44; Pereira et al., J Struct Biol. (2010), 172(3): 372-9; Poidevin et al., "Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction", AppI Environ Microbiol. (2013), 79(14): 4220-9.

Endoglycoceramidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,795,765; or US patent application publication No. US 2009/0170155 A1.

Endo-inulinase. Polypeptides of the present invention having this activity can be characterized for example as described in Vandamme et al., FEBS Open Bio. (2013), 3: 467^72; US patent No. 8,309,079.

Endo-polygalacturonase. Polypeptides of the present invention having this activity can be characterized for example as described in European patent application publication Nos. EP1614748 A1 and EP1114165 A1.

Endo-polygalacturonase. Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 1994/014952 Al ; or in European patent application publication No. EP1614748 A1.

Endo-rhamnogalacturonase GH28. Polypeptides of the present invention having this activity can be characterized for example as described in Sprockett et al., Gene (2011), 479(1-2): 29-36; or An et al., Carbohydrate Research (1994), 264(1): 83-96.

Exo-1,3-beta-galactanase GH43. Polypeptides of the present invention having this activity can be characterized for example as described in Ichinose et al., AppI Environ Microbiol. (2006), 72(5): 3515-3523.

Exo-1,3-beta-glucanase GH17. Polypeptides of the present invention having this activity can be characterized for example as described in Wojtkowiak et al., Acta Crystallogr D Biol Crystallogr. (2013) 69(Pt 1):52-62; Tao et al., Gene (2013), 527(1 ):154-60. Exo-1 ,3-beta-glucanase Polypeptides of the present invention having this activity can be characterized for example as described in O'Connell et al., Appi Microbiol Biotechnol. (2011), 89(3): 685-96; or Santos et al., J Bacteriol. (1979), 139(2): 333-338.

Exo-1,4-beta-xylosidase. Polypeptides of the present invention having this activity can be characterized for example as described in La Grange et al., Applied and Environmental Microbiology (2001), 67(12): 5512-5519.

Exo-arabinanase. Polypeptides of the present invention having this activity can be characterized for example as described in Tatsuji Sakamoto and Thibault, Appi Environ Microbiol. (2001 ), 67(7): 3319-3321.

Exo-beta-D-glucosaminidase. Polypeptides of the present invention having this activity can be characterized for example as described in Honda et al., Glycobiology. (2011), 21 (4): 503-11 ; Li et al., Carbohydr Res. (2009), 344(8): 1046-9; Fukamizo et al., Glycobiology. (2006), 16(11): 1064-72; Nogawa et al., Appi Environ Microbiol. (1998), 64(3): 890-5; Jung et al., Protein Expr Purif. (2006), 45(1): 125-31.

Exoglucanase. Polypeptides of the present invention having this activity can be characterized for example as described in Creuzet et al., FEMS Microbiology Letters (1983), 20(3): 347-350; or Kruus et al., Joumai of Bacteriology (1995), 177(6): 1641-1644.

Exo-glucosaminidase GH2. Polypeptides of the present invention having this activity can be characterized for example as described in Tanaka et al., Journal of Bacteriology (2003), 185(17): 5175-5181.

Exo-inulinase. Polypeptides of the present invention having this activity can be characterized for example as described in Kulminskaya et al., Biochimica et Biophysica Acta (2003), 1650(1-2):22-9; Pessoni et al., Mycologia. (2007), 99(4):493-503.

Exo-polygalacturonase Polypeptides of the present invention having this activity can be characterized for example as described in Dong and Wang, BMC Biochem. (2011), 12: 51.

Exo-rhamnogalacturonase GH28. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,811 ,291.

Expansin. Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 2005/030965 A2; or in US patent No. 7,001 ,743.

Expansin-like protein 1. Polypeptides of the present invention having this activity can be characterized for example as described in Lee et al., Molecules and Cells (2010), 29(4): 379-85.

Extracellular endo-alpha-(1->5)-L-arabinanase 1. Polypeptides of the present invention having this activity can be characterized for example as described in Inacio and de Sa-Nogueira, J Bacteriol. (2008), 190(12):4272- 80.

Extracellular metalloproteinase 10. Polypeptides of the present invention having this activity can be characterized for example as described in Almeida et al., Parasitol Res. (2003), 89(4); Shibata et al., J Biol Chem. (2000), 275(12):8349-54; Kim and Kim, Can J Microbiol. (1994), 40(2):120-6.

Feruloyl esterase. Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 2009/076122 A1.

Galactanase GH5. Polypeptides of the present invention having this activity can be characterized for example as described in lchinose et al., Applied and Environmental Microbiology (2008), 74(8): 2379-2383.

Galacturan 1,4-alpha-galacturonidase C. Polypeptides of the present invention having this activity can be characterized for example as described in Dong and Wang, BMC Biochem. (2011), 12:51.

Glucan 1,3-beta-glucosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Boonvitthya et al., Biotechnol Lett (2012), 34(10): 1937-43.

Glucan endo-1,3-beta-glucosidase Polypeptides of the present invention having this activity can be characterized for example as described in Sperisen et al., Proc Natl Acad Sci USA (1991), 88(5):1820-4.

Glucan endo-1,6-beta-glucosidase B. Polypeptides of the present invention having this activity can be characterized for example as described in Fayad et al., Appi Microbiol Biotechnol. (2001), 57(1 -2): 117-23. Gluconolactonase. Polypeptides of the present invention having this activity can be characterized for example as described in Kondo et al., Proc Natl Acad Sci USA (2006), 103(15):5723-8; and Tarighi et al., Microbiology (2008), 154(Pt 10):2979-90.

Glucose oxidase. Polypeptides of the present invention having this activity can be characterized using a commercially available kit such as Amplex® Red Glucose/Glucose Oxidase Assay Kit (Cat. No. A22189, Life Technologies).

Glucose-6-phosphate 1-epimerase. Polypeptides of the present invention having this activity can be characterized for example as described in Wurster and Hess, Methods Enzymol. (1975), 41 : 488-93.

Glucosylceramidase Polypeptides of the present invention having this activity can be characterized for example as described in Vaccaro et al., Eur J Biochem. (1985), 146(2): 315-21 ; Vaccaro et al., Enzyme. (1989), 42(2): 87-97; Vaccaro et al., Clin Chim Acta. (1982), 118(1 ): 1-7.

Glycosidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 8,119,383.

Hephaestin-like protein 1. Polypeptides of the present invention having this activity can be characterized for example as described for oxioreductases.

Hexosaminidase. Polypeptides of the present invention having this activity can be characterized for example as described in Wendeler and Sandhoff, Glycoconj J. (2009), 26(8):945-952.

Hydrophobin. Polypeptides of the present invention having this activity can be characterized for example as described in Bettini et al., Canadian Journal of Microbiology (2012), 58(8): 965-972; or Niu et al., Amino Acids. (2012), 43(2)763-71.

Invertase. Polypeptides of the present invention having this activity can be characterized for example as described in Bacon, J.S.D., Methods in Enzymology (1955), Volume I, 258-262; Lever, M. Analytical Biochemistry (1972), Volume 47, 273-279; Us patent No. US 5,665,579.

Iron transport multicopper oxidase FET3. Polypeptides of the present invention having this activity can be characterized for example as described in Askwith et al., Cell (1994), 76: 403-10; or De Silva et al., J. Biol. Chem. (1995) 270: 1098-1101.

Laccase. Polypeptides of the present invention having this activity can be characterized for example as described in Dedeyan et al., Appl Environ Microbiol. (2000), 66(3): 925-929.

Lactonase. Polypeptides of the present invention having this activity can be characterized for example as described in Khersonsky and Tawfik, ChemBioChem (2006), 7(1): 49-53; or Chow et al„ J Biol Chem. (2010) 285(52):40911 -20.

Laminarinase GH55. Polypeptides of the present invention having this activity can be characterized for example as described in Ishida et al., J Biol Chem. (2009), 284(15): 10100-10109; or Kawai et al., Biotechnol Lett. (2006), 28(6): 365-71.

L-Ascorbate oxidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent Nos. 5,612,208 and 5,180,672.

L-carnitine dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Aurich et al., Biochim Biophys Acta. (1967), 139(2): 505-7; or US patent No. 5,156,966.

Leucine aminopeptidase 1. Polypeptides of the present invention having this activity can be characterized for example as described in Beattie et al., Biochem. J. (1987), 242: 281-283.

Licheninase (beta-D-glucan 4-glucanohydrolase). Polypeptides of the present invention having this activity can be characterized for example as described in Tang et al., J Agric Food Chem. (2012), 60(9): 2354-61.

Lipase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent Nos. 7,662,602 and 7,893,232. L-lactate dehydrogenase A. Polypeptides of the present invention having this activity can be characterized for example as described in Yeswanth et al., Anaerobe (2013), 24:43-8; Xia et al„ Mol Biol Rep. (2011 ),

38(3): 1853-60.

Loosenin. Polypeptides of the present invention having this activity can be characterized for example as described in Quiroz-Castaneda et al., Microbial Cell Factories (2011), 10:8.

L-sorbosone dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Shinjoh et al., Applied and Environment Microbiology (1995), 61 (2): 413-420.

Lysophospholipase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,965,422.

Lysozyme. Polypeptides of the present invention having this activity can be characterized for example as described in EnzChek® Lysozyme Assay Kit (cat. No. E22013, Life Technologies); Shugar, D. Biochimica et Biophysica Acta (1952), 8: 302-309.

Metallocarboxypeptidase. Polypeptides of the present invention having this activity can be characterized for example as described in Tayyab et al., J Biosci Bioeng. (2011 ), 111 (3): 259-65; or Song et al., J Biol Chem. (1997), 272(16): 10543-50.

Methylenetetrahydrofolate dehydrogenase [NAD(+)]. Polypeptides of the present invention having this activity can be characterized for example as described in Wohlfarth et al., J Bacteriol. (1991), 173(4): 1414— 1419.

Mixed-link glucanase. Polypeptides of the present invention having this activity can be characterized for example as described in Clark et al., Carbohydr Res. (1978), 61 : 457-477.

Monooxygenase. Polypeptides of the present invention having this activity can be characterized for example as described in Haghbeen and Tan, Anal Biochem. (2003), 312(1 ): 23-32.

Mucorpepsin. Polypeptides of the present invention having this activity can be characterized for example as described in Baudy et al., FEBS Lett. (1988), 235: 271-274.

Multicopper oxidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0094335 A1.

Mutanase. Polypeptides of the present invention having this activity can be characterized for example as described in Pleszczynska, Biotechnol Lett. (2010), 32(11): 1699-1704; or WO 1998/000528 A1.

N-acetylglucosaminidase GH18. Polypeptides of the present invention having this activity can be characterized for example as described in Murakami et al., Glycobiology (2013), e-pub: Feb.22, PMID:

23436287; or in US patent application publication No. US20120258089 A1.

N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D. Polypeptides of the present invention having this activity can be characterized for example as described in Guo et al., J Lipid Res. (2013),

54(11):3151-7.

NADPH-cytochrome P450 reductase. Polypeptides of the present invention having this activity can be characterized for example as described in Guengerich et al., Nat Protoc. (2009), 4(9): 1245-51.

NADPH-dependent methylglyoxal reductase GRE2. Polypeptides of the present invention having this activity can be characterized for example as described in Murata et al., Eur. J. Biochem. (1985), 151 (3): 631-636; Johnston et al., Yeast. (2003), 20 (6): 545-554.

Non-hemolytic phospholipase C. Polypeptides of the present invention having this activity can be characterized for example as described in Weingart and Hooke, Curr Microbiol. (1999), 38(4): 233-8; Korbsrisate et al., J Clin Microbiol. (1999), 37(11 ): 3742-5. Oxidase. Polypeptides of the present invention having this activity can be characterized for example using a number of commercially available kits [e.g., Amplex® Red Galactose/Galactose Oxidase Kit (A22179) and Amplex® Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes/lnvitrogen); Cytochrome C Oxidase Assay Kit (Cat. No. CYTOCOX1-1 KT; Sigma-Aldrich); Xanthine Oxidase Assay Kit (ab102522, Abeam); Lysyl Oxidase Activity Assay Kit (ab112139, Abeam); Glucose Oxidase Assay Kit (ab138884, Abeam); Monoamine oxidase B (MAOB) Specific Activity Assay Kit (ab109912, Abeam)].

Oxidoreductase. Polypeptides of the present invention having this activity can be characterized for example as described in Hommes et al., Anal Chem. (2013), 85(1 ): 283-291.

Para-nitrobenzyl esterase. Polypeptides of the present invention having this activity can be characterized for example as described in Moore and Arnold, Nat Biotechnol. (1996), 14(4): 458-67.

Pectate lyase. Polypeptides of the present invention having this activity can be characterized for example as described in Wang et al., BMC Biotechnology (2011 ), 11 : 32.

Pectin methylesterase. Polypeptides of the present invention having this activity can be characterized for example as described in PCT application publication No. WO 1997/031102 A1.

Pectinesterase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 5,053,232.

Penicillopepsin. Polypeptides of the present invention having this activity can be characterized for example as described in Cao et al., Protein Sci. (2000), 9(5): 991-1001 ; or Hofmann et al., Biochemistry. (1984), 14;23(4): 635-43.

Peroxidase. Polypeptides of the present invention having this activity can be characterized for example using a number of commercially available kits [e.g., Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/lnvitrogen); Peroxidase Activity Assay Kit (Cat. No. K772-100; BioVision); QuantiChrom™ Peroxidase Assay Kit (Cat. No. DPOD-100, BioAssay Systems].

Peroxisomal hydratase-dehydrogenase-epimerase. Polypeptides of the present invention having this activity can be characterized for example as described in Nuttley et al., Gene. (1988), 69(2):171-80.

Phenol 2-monooxygenase (phenol hydroxylase). Polypeptides of the present invention having this activity can be characterized for example as described in Nakagawa and Takeda, Biochim. Biophys. Acta. (1962), 62 (2): 423-6; Neujahr and Gaal, Eur. J. Biochem. (1973), 35(2): 386-400; Neujahr and Gaal, Eur. J. Biochem. (1975), 58(2): 351-7; Kirchner et al., J Biol Chem. (2003), 278(48): 47545-53.

Phospholipase C. Polypeptides of the present invention having this activity can be characterized for example using commercially available kits (Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit, Molecular Probes/lnvitrogen).

Polyphenol oxidase 1. Polypeptides of the present invention having this activity can be characterized for example as described in Tao et al., J Agric Food Chem. (2013), 61 (51): 12662-9; Dawson and Magee, Methods in Enzy ology ll (1955), 817-821 ; Marumo and Waite, Biochim. Biophys. Acta (1986), 872: 98-103.

Polysaccharide lyase. Polypeptides of the present invention having this activity can be characterized for example as described in Macdonald and Berger, "A polysaccharide lyase from Stenotrophomonas maltophilia with unique, pH-regulated substrate specificity.", J. Biol Chem. (2013); Cordula et al., "On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+", Mol Biosyst. (2013); or in PCT application publication No. WO 2013007706 A1.

Polysaccharide monooxygenase. Polypeptides of the present invention having this activity can be characterized for example as described in KittI et al., Biotechnol Biofuels. (2012), 5(1):79, Phillips et al., ACS Chem Biol (2011), 6(12): 1399-1406, Wu et al., J. Biol. Chem (2013), 288(18): 12828-39. Polysaccharide monooxygenases, sometimes referred to functionally as "cellulase-enhancing proteins", generally belong the enzyme class GH61 and are reported to cleave polysaccharides with the insertion of oxygen. Protease or peptidase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2005/0010037 A1 ; using the EnzChek® Peptidase/Protease Assay Kit (Cat. No. E33758, Life Technologies).

Putative exoglucanase type C (1 ,4-beta-cellobiohydrolase; beta-glucancellobiohydrolase;

exocellobiohydrolase I). Polypeptides of the present invention having this activity can be characterized for example as described in Dai et al., Applied Biochemistry and Biotechnology (1999), 79, Issue 1-3: 689-699.

Pyranose dehydrogenase. Polypeptides of the present invention having this activity can be characterized for example as described in Staudigl et al., Biomolecules (2013), 3: 535-552; Tan et al, PLoS One. (2013), 8(1):e53567; Kujawa et al, FEBS J. (2007), 274(3): 879-94.

Rhamnogalacturonan acetylesterase. Polypeptides of the present invention having this activity can be characterized for example as described in Molgaard et al, Sfrucfu/-e(2000), 8(4):373-83; or Kauppinen et al, J Biol Chem. (1995), 270(45):27172-8.

Rhamnogalacturonan endolyase. Polypeptides of the present invention having this activity can be characterized for example as described in Azadi et al, Glycobiology. (1995), 5(8): 783-9.

Rhamnogalacturonan lyase. Polypeptides of the present invention having this activity can be characterized for example as described in Mutter et al. Plant Physiol. (1998), 117: 153-163; or de Vries, Appl. Microbiol Biotechnol. (2003), 61 : 10-20.

Rhamno-galacturonate lyase. Polypeptides of the present invention having this activity can be characterized for example as described in Jensen et al, J Mol Biol. (2010), 404(1 ): 100-11.

Rhizopuspepsin-3. Polypeptides of the present invention having this activity can be characterized for example as described in Chen et al, J Agric Food Chem. (2009), 57(15):6742-7; Flentke et al. Protein Expr Purif. (1999), 16(2):213-20.

Rodlet protein. Polypeptides of the present invention having this activity can be characterized for example as described in Yang et al, Biopolymers (2013), 99(1 ): 84-94.

Saccharopine dehydrogenase [NADP(+), L-glutamate-forming] Polypeptides of the present invention having this activity can be characterized for example as described in Kumar et al. Arch Biochem Biophys. (2012), 522(1 ):57-61 ; Ekanayake et al. Arch Biochem Biophys. (2011), 514(1-2):8-15.

Serine-type carboxypeptidase F. Polypeptides of the present invention having this activity can be characterized for example as described in US patent No. 6,379,913.

Short-chain dehydrogenase reductase 2a. Polypeptides of the present invention having this activity can be characterized for example as described in Bijtenhoorn et al, PLoS One. (2011 ), 6(10):e26278; Polizzi et al, Chem Commun (Camb). (2007), 18:1843-5.

Subtilisin-like protease 6. Polypeptides of the present invention having this activity can be characterized for example as described in Dang et al, J Invertebr Pathol. (2013), 112(2): 166-74; Acevedo et al, J Appl Microbiol. (2013), 114(2):352-63.

Swollenin. Polypeptides of the present invention having this activity can be characterized for example as described in Jager et al, Biotechnol Biofueis. (2011 ), 4: 33; or Saloheimo et al., Eur J Biochem. (2002), 269(17): 4202-11.

Tripeptidyl-peptidase sedl . Polypeptides of the present invention having this activity can be characterized for example as described in Du et al, Biol Chem. (2001), 382(12):1715-25; Hilbi et al, Biochim Biophys Acta. (2002), 1601(2):149-54; Renn et al, J Biol Chem. (1998), 273(30): 19173-82.

Tyrosinase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2011/0311693 A1 ; Duckworth and Coleman, J. Biol. Chem. (1970) , 245: 1613-1625; Park et al, J Protein Chem. (2003), 22(5): 473-80.

Unsaturated rhamnogalacturonyl hydrolase YteR. Polypeptides of the present invention having this activity can be characterized for example as described in Itoh et al, Biochem Biophys Res Commun. (2006), 347(4): 1021-9; or Itoh et al, J Mol Biol. (2006), 360(3): 573-85. Versatile peroxidase. Polypeptides of the present invention having this activity can be characterized for example as described in Lankinen et al., Appl Microbiol Biotechnol. (2005), 66(4): 401-7; Banci et al., J Biol Inorg Chem. (2003), 8(7): 751-60; Perez-Boada et al., J Mol Biol. (2005), 354(2): 385-402.

Xylan alpha-1 ,2-glucuronidase. Polypeptides of the present invention having this activity can be characterized for example as described in Ishihara, M. and Shimizu, K., "alpha-(1->2)-Glucuronidase in the enzymatic saccharification of hardwood xylan: Screening of alpha-glucuronidase producing fungi." Journal Mokuzai Gakkaishi, (1988) 34: 58-64.

Xylanase. Polypeptides of the present invention having this activity can be characterized for example as described in US patent application publication No. US 2012/0028306 A1 ; US patent No. 7,759,102; PCT application publication No. WO 2006/078256 A2; Chen et al., Agric. Biol. Chem. (1986), 50: 1183-1194; Lever, M., Analytical Biochemistry (1972), 47: 273-279.

Xyloglucanase. Polypeptides of the present invention having this activity can be characterized for example as described in Master et al., Biochem. (2008), 411 (1 ): 161-170; Ariza et al., J Biol Chem. (2011), 286(39): 33890- 900; Qi et al., Biochemistry (Mosc). (2013), 78(4): 424-30; US patent No. 6,815,192.

Xyloglucan-specific endo-beta-1 ,4-glucanase A. Polypeptides of the present invention having this activity can be characterized for example as described in European patent application publication No. EP0972016 B1 ; in US patent No. 6,077,702; Damasio et al, Biochim Biophys Acta. (2012), 1824(3): 461-7; or Wong et al, Appl Microbiol Biotechnol. (2010), 86(5): 1463-71.

Xylosidase/arabinosidase. Polypeptides of the present invention having this activity can be characterized for example as described in Whitehead and Cotta, Curr Microbiol. (2001), 43(4): 293-8; or Xiong et al. Journal of Experimental Botany (2007), 58(11): 2799-2810.

Example 6: General Molecular Biology Procedures

[00242] Standard molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, £ coli transformation, etc, were performed as described by Sambrook et al, 1989, (Molecular cloning: A laboratory manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and Innes et al. (1990) PCR protocols, a guide to methods and applications, Academic Press, San Diego, edited by Michael A. Innis et al). Primers were prepared by IDT (Integrated DNA Technologies). Sanger DNA sequencing was performed using an Applied Biosystem's 3730x1 DNA Analyzer technology at the Innovation Centre (Genome Quebec), McGill University in Montreal.

Example 7: Construction of pGBFIN49 expression plasmids

[00243] Genes of interest were cloned into the expression vector pGBFIN-49. This vector is a derivative of pGBFIN-41 that contains the A. niger glaA promoter, A. niger JrpC terminator, A. nidulans gpdA promoter, gene encoding the pheomycin resistance gene, A. niger glaA terminator and an E. coli backbone. Figure 1 represents a schematic map of pGBFIN-49 and the complete nucleotide sequence is presented as SEQ ID NO: 1543. Details of the construction of pGBFIN-49 are as follows:

1 TtrpC terminator PCR amplification (0.7kb):

[00244] TtrpC terminator was PCR amplified using purified pGBFIN33 plasmid as a template. The following primers and PCR program were used:

Primer-3 : 5 ' -GTCCGTCGCCGTCCTTCAccgccggtccgacg-3 ' Primer-4 : 5 ¹ -GCGGCCGGCGTATTGGGTGttacggagc-3 ' [00245] Primer-4 is entirely specific to the TtrpC 3' end. Primer-3 was designed to suit the LIC cloning strategy but also to keep the TtrpC sequence as close to the original sequence. To do so, five adenines were replaced by thymines (underlined).

PCR master mix:

pGBFIN33 1 L (5-10 ng)

Primer-3 (10 mM) 1 \il

Primer-4 (10 mM) 1 \ii

dNTPs (2 mM) 5 μΙ_

HF Buffer (5x) 10 μί

Phusion DNS pol. 0.5 L

Nuclease-free water 31.5 ML

Total 50 μί

[00246] PCR program: 1 x 98°C, 2 min; 25 x (98°C, 30 sec; 68°C, 30 sec; 72°C, 1 min); 72°C, 7 min.

[00247] Reaction conditions: 5 pL of the PCR reaction was separated by electrophoresis on 1.0% agarose gel and the remaining was purified using QIAEX II™ gel Extraction kit (QIAGEN) and resuspended in nuclease- free water.

2. PGBFIN41 vector PCR amplification (8.3kb):

[00248] Vector backbone was PCR amplified using pGBFIN41 as a template. Primers were designed outside of the ccdA region (not included in pGBFIN49). The following primers and PCR program were used:

Primer-2 : 5 ' -CACCCAATACGCCGGCCGCgcttccagacagctc-3 ' Primer-lC : 5 ' -GGTGTTTTGTTGCTGGGGAtgaagctcaggctctcagttgcgtc-3 '

[00249] Primer-2 contains a pgpdA-specific region and an extra sequence specific to TtrpC 3' end (also included in Primer-4). Prime C was designed to suit the LIC cloning strategy but also to keep PgalA region as close to the original sequence. To do so, three thymines were replaced by adenines (underlined).

PCR master mix:

pGBFIN41 1 ML (50 ng)

Primer-2 (10 mM) 1 ML

PrimeMC (10 mM) 1 ML

dNTPs (2 mM) 5 ML

HF Buffer (5x) 10 μί

Phusion DNS pol. 0.5 ML

DMSO 1 μί

Nuclease-free water 30.5 μί

Total 50 μί

[00250] PCR program: 1 x 98°C, 3 min; 10x (98°C, 30 sec ; 68°C, 30 sec, 72°C, 5 min); 20 x (98°C, 30 sec, 68°C, 30 sec, 72°C, 5 min + 10 sec/cycle); 72°C, 10 min.

[00251] Reaction conditions: 5 ML of the PCR reaction was separated on a 0.5% agarose gel and remaining was purified using QIAEX II™ gel Extraction kit (QIAGEN) and resuspended in nuclease-free water.

3 _; PGBFIN41 + TtrpC overlap-extension PCR: [00252] Overlap-extension / Long range PCR was performed to: a) fuse the two PCR pieces together; b) add an Sfol restriction site to re-circularize the vector. No primers were used in the overlap-extension stage. Primer-11 and Primer-12 were used for the long range PCR reaction.

Primer-ll: 5 ' -CACCGGCGCCGTCCGTCGCCGTCCTTC -3' Primer-12: 5 ' -ACGGCGCCGGTGTTTTGTTGCTGGGGATG -3'

[00253] Primer-11 is specific to the LIC tag located on the TtrpC terminator, while Primer-12 is specific to the LIC tag located on the PglaA region. The Sfol restriction site sequence is underlined above.

[00254] A standard PCR master mix was prepared to perform overlap-extension PCR using pGBFIN41 and TtrpC purified PCR products as templates. No primers were added.

Overlap-extension master mix:

TtrpC 1 μί

pGBFIN41 9 μί

Buffer GC (5x) 10 pL

dNTPs (2 mM) 5 μί

Phusion DNA pol. 0.5 μί

Nuclase-free water 24.5 \}L

PGBFIN41 50 pL

[00255] PCR program - overlap (no primers): 1x 98°C, 2 min; 5x (98°C, 15 sec; 58°C, 30 sec; 72°C, 5 min), 5x (98°C, 15 sec; 63°C, 30 sec; 72°C, 5 min), 5x (98°C, 15 sec; 68°C, 30 sec; 72°C, 5 min); 72°C, 10 min.

[00256] The overlap-extension PCR product was then, purified on QIAEX II™ column and 5 pL of the purified reaction was used as template DNA for Long range PCR step with Primers-11 and -12.

PCR master mix:

Overlap product 5 ML

Primer-11 (10mM) 1 pL

Primer-12 (10mM) 1 μί

dNTPs (2mM) 5 pL

HF Buffer (5x) 10 pL

Phusion DNA pol. 0.5 pL

DMSO 1 pL

Nuclease-free water 26.5 pL

pGBFIN41 50 pL

[00257] PCR program - Long range: 1x 98°C, 3 min; 10x (98°C, 30 sec ; 68°C, 30 sec ; 72°C, 5 min); 20 x (98°C, 30 sec ; 68°C, 30 sec ; 72°C, 5 min + 10 sec/cycle); 72°C, 10 min.

[00258] Reaction conditions: 5 pL of the PCR reaction was separated on 0.5% agarose gel and remaining was purified using QIAEX II™ gel Extraction kit and resuspended in nuclease-free water. Then, Sfol digestion was performed and digested product was purified using QIAEX II gel extraction kit follow the procedure as described by the manufacturer.

4. Ligation:

[00259] 100 ng of the purified digested fragment was ligated to itself using 1 pL of T4 DNA Ligase (New England Biolabs, M0202), and incubated at 16°C overnight. Enzyme inactivation was performed at 65°C for 10 minutes. Then, 10 pL of ligation product was transformed in DH5 E. coli competent cells and plated on 2xYT agar containing 100 ug/mL ampicillin. DNA extraction was performed on single colonies the next day. Restriction analysis and sequencing were done to confirm the structure.

Example 8: Cloning of Amorphotheca resinae, Rhizomucor pusillus, and Calcarisporiella thermophila genes in E. coli

[00260] Cloning genes of interest in the pGBFIN-49 expression vector was performed using the Ligation- independent cloning (LIC) method according to Aslanidis, C, de Jong, P. (1990) Nucleic Acids Research Vol. 18 No. 20, 6069-6074.

[00261] Coding sequences from genes of interest were amplified by PCR using primers containing LIC tags, which are homologous to Pg/a and TrpC sequences in the pGBFIN-49 cloning vector fused to sequences homologous to the coding sequences of the gene of interest, and either genomic DNA or cDNA as template. Primers have the following sequences:

Forward primer: 5 -CCCCAGCAACAAAACACCTCAGCAATG...15-20 nucleotides specific to each gene to be cloned

Reverse primer: 5 -GAAGGACGGCGACGGACTTCA...15-20 nucleotides specific to each gene to be cloned

PCR mix consists of following components:

Template (gDNA or cDNA) 1-10 ng/ L 1 μί

5X Phusion HF Buffer (Finnzymes™) 10 pL

2 mM dNTPs 5 \ii

LIC primer (F+R) mix 10 mM 0.5 μί

Phusion DNA Polymerase (Finnzymes™) 0.5 \ii

DMSO 1.5 ML

H ₂ Q 31.5 ML

TOTAL 50 pL

[00262] PCR amplification was carried out with following conditions:

3-step protocol

Cycle step Cycles

Temp Time

Initial denaturation 98°C 30 s 1

Denaturation 98°C 10 s

Annealing 58°C 30 s 10

Extension 72°C 30 s

Denaturation 98°C 10 s

Annealing 68°C 30 s 20

Extension 72°C 30 s

Final extension 70°C 10 min 1

End of PCR storage 4°C hold 1 [00263] Following PCR, 90 μΙ_ milliQ™ water was added to each sample and the mix was purified using a Multiscreen PCRge Filter Plate (Millipore) according to manufacturer's instructions. The PCR product was eluted from the filter in 25 pL 10 mM Tris-HCI pH 8.0.

[00264] Expression vector pGBFIN-49 was PCR amplified using primers with following sequences:

Forward primer: 5 ' -GTCCGTCGCCGTCCTTCACCG-3 ' Reverse primer: 5' -GGTGTTTTGTTGCTGGGGATGAAGC-3'

(Primers are located at either site of the S/ l restriction site.)

PCR mix consists of following components:

pGBFIN-49 plasmid DNA (10 ng/ μ|_) 2 \iL

5X Phusion HF Buffer (Finnzymes™) 20 pL

2 mM dNTPs 10 pL

LIC Primer mix (F+R) 10 mM 2 pL

Phusion DNA Polymerase (Finnzymes™) 1.5 pL

DMSO 3 pL

H ₂ Q 61.5 ML

TOTAL 100 pL

[00265] PCR amplification was carried out with following conditions:

[00266] Following PCR, 1 il of Dpn\ was added to the PCR mix and digestion was performed overnight at 37°C. Digested PCR product was purified using the Qiaquick™ PCR purification kit (Qiagen) according to manufacturer's instructions.

[00267] Obtained PCR fragments were treated with T4 DNA polymerase in the presence of dTTP to create single stranded tails at the ends of the PCR fragments. The single stranded tails of the PCR fragment are complementary to those of the vector, thus permitting non-covalent bi-molecular associations, e.g., circularization between molecules.

[00268] The reaction mix of the T4 DNA polymerase treatment of the pGBFIN-49 PCR fragment consisted of the following components:

Purified pGBFIN-49 PCR fragment 600 ng

10X Neb Buffer 2 2 pL

25 mM dTTP 2 pL

DTMOO pM 0.8 pL

T4 DNA Polymerase 311/ pL 1 pL

H ₂ 0 Up to 20 pL TOTAL 20 pL

[00269] The reaction mix of T4 DNA polymerase treatment of the Gene of Interest (GOI) PCR fragment consisted of the following components:

Purified GOI PCR 5 pL

10X NEB Buffer 2 2 pL

25 mM dATP 2 pL

DTT 100 pM 0.8 pL

T4 DNA Polymerase 311/ pL 1 pL

H ₂ 0 9.2 pL

TOTAL 20 pL

[00270] Reaction conditions were as follows:

[00271] Following T4 DNA polymerase treatment, 2 L of pGBFIN-49 vector and 4 pL of the GOI were mixed and incubated at room temperature allowing annealing of GOI fragment with pGBFIN-49 vector fragment. The bi-molecular forms are used to transform E. coli. Plasmid DNA of resulting transformants was isolated and verified by sequence analyses for correct amplification and cloning of the gene of interest.

Example 9: Transformation of Amorphotheca resinae, Rhizomucor pusillus, and Calcarisporiella thermophila gene expression cassettes into A. niger

[00272] As host strain for enzyme production, A. niger GBA307 was used. Construction of A. niger GBA307 is described in WO 2011/009700.

[00273] Transformation of A. niger was performed essentially according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J & Hynes, M. (1985) EMBO 1, 4, 475-479 with the following modifications:

Spores were grown for 16-24 hours at 30°C in a rotary shaker at 250 rpm in Aspergillus minimal medium. Aspergillus minimal medium contains per liter: 6 g NaN0 ₃ ; 0.52 g KCI; 1.52 g KH ₂ P0 ₄ ; 1.12 ml 4 M KOH; 0.52 g MgS0 ₄ -7H ₂ 0; 10 g glucose; 1 g casamino acids; 22 mg ZnS0 ₄ -7H 0; 11 mg H ₃ B0 ₃ ; 5 mg FeS0 ₄ -7H ₂ 0; 1.7 mg CoCI ₂ -6H ₂ 0; 1.6 mg CuS0 -5H ₂ 0; 5 mg MnCI ₂ -2H ₂ 0; 1.5 mg Na ₂ Mo04'2 H ₂ 0; 50 mg EDTA; 2 mg riboflavin; 2 mg thiamine-HCI; 2 mg nicotinamide; 1 mg pyridoxine-HCI; 0.2 mg panthotenic acid; 4 pg biotin; 10 ml Penicillin (5000IU/mL/Streptomycin (5000 UG/mL) solution (Invitrogen);

Glucanex 200G (Novozymes) was used for the preparation of protoplasts;

- After protoplast formation (2-3 hours) 10 mL TB layer (per liter: 109.32 g Sorbitol; 100 mL 1 M Tris- HCI pH 7.5) was pipetted gently on top of the protoplast suspension. After centrifugation for 10 min at 4330 x g at 4°C in a swinging bucket rotor, the protoplasts on the interface were transferred to a fresh tube and washed with STC buffer (1.2 M Sorbitol, 10 mM Tris-HCI pH 7.5, 50 mM CaCI ₂ ). The protoplast suspension was centrifuged for 10 min at 1560 x g in a swinging bucket rotor and resuspended in STC-buffer at a concentration of 10 ^s protoplasts/mL;

To 200 μί of the protoplast suspension, 20 μί. ATA (0.4 M Aurintricarboxylic acid), the DNA dissolved in 10 μί in TE buffer (10 mM Tris-HCI pH 7.5, 0.1 mM EDTA), 100 μΐ of a PEG solution (20% PEG 4000 (Merck), 0.8M sorbitol, 10 mM Tris-HCI pH 7.5, 50 mM CaCI ₂ ) was added;

After incubation of the DNA-protoplast suspension for 10 min at room temperature, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCI pH7.5, 50 mM CaC ) was added slowly, with repeated mixing of the tubes. After incubation for 20 min at room temperature, suspensions were diluted with 5 ml 1.2 M sorbitol, mixed by inversion and centrifuged for 10 min at 2770 x g at room temperature.

The protoplasts were resuspended gently in 1 mL 1.2 M sorbitol and plated onto selective regeneration medium consisting of Aspergillus minimal medium without riboflavin, thiamine.HCI, nicotinamide, pyridoxine, panthotenic acid, biotin, casamino acids and glucose, supplemented with 150 pg/mL Phleomycin (Invitrogen), 0.07 M NaNC>3, 1 M sucrose, solidified with 2% bacteriological agar #1 (Oxoid, England). After incubation for 5-10 days at 30°C, single transformants were isolated on PDA (Potato Dextrose Agar (Difco) supplemented with 150 pg/mL Phleomycin in 96 wells MTP. After 5-7 days growth at 30°C single transformants were used for MTP fermentation.

Example 10: Aspergillus niger microtiter plate fermentation

[00274] 96 wells microtiter plates (MTP) with sporulated Aspergillus niger strains were used to harvest spores for MTP fermentations. To do this, 100 pL water was added to each well and after resuspending the mixture, 40 pL of spore suspension was used to inoculate 2 mL A.niger medium (70 g/L glucose-H ₂ 0, 10 g/L yeast extract, 10 g/L (NH ) ₂ S0 , 2 g/L K ₂ S0 ₄ , 2 g/L KH ₂ P0 ₄ , 0.5 g/L MgS0 -7H ₂ 0, 0.5 g/L ZnS0 ₄ -7H ₂ 0, 0.2 g/L CaCI ₂ , 0.01 g/L MnS0 ₄ -7H ₂ 0, 0.05 g/L FeS0 -7H ₂ 0, 0.002 Na ₂ Mo0 ₄ -2H ₂ 0, 0.25 g/L Tween™-80, 10 g/L citric acid, 30 g/L MES; pH 5.5 adjusted with 4 M NaOH) in a 24 well MTP. In the MTP fermentations for strains expressing GH61 proteins (e.g., polysaccharide monooxygenases), 30 μΜ CuS0 ₄ was included in the media. The MTP's were incubated in a humidity shaker (Infers) at 34°C at 550 rpm, and 80% humidity for 6 days. Plates were centrifuged and supernatants were harvested.

Example 11 : Aspergillus niger shake flask fermentation

[00275] Approximately 1x10 ⁶ - 1x10 ⁷ spores were inoculated in 20 mL pre-culture medium containing Maltose 30 g/L; Peptone (aus casein) 10 g/L; Yeast extract 5 g/L; KH ₂ P0 1 g/L; MgS0 ₄ -7H ₂ 0 0.5 g/L; ZnCI ₂ 0.03 g/L; CaCI ₂ 0.02 g/L; MnS0 ₄ -4H ₂ 0 0.01 g/L; FeS0 ₄ -7H ₂ 0 0.3 g/L; Tween™-80 3 g/L; pH 5.5. After growing overnight at 34°C in a rotary shaker, 10-15 mL of the growing culture was inoculated in 100 mL main culture containing Glucose'H ₂ 0 70 g/L; Peptone (aus casein) 25 g/L; Yeast extract 12.5 g/L; K ₂ S0 2 g/L; KH ₂ P0 ₄ 1 g/L; MgS0 ₄ -7H ₂ 0 0.5 g/L; ZnCb 0.03 g/L; CaCI ₂ 0.02 g/L; MnS0 ₄ -1 H ₂ 0 0.009 g/L; FeSCy7H ₂ 0 0.003 g/L; pH 5.6. Note: for GH61 (e.g., polysaccharide monooxygenase) enzymes the culture media were supplemented with 10 μΜ CuS0 ₄ .

[00276] Main cultures were grown until all glucose was consumed as measured with Combur Test N strips (Roche), which was the case mostly after 4-7 days of growth. Culture supernatants were harvested by centrifugation for 10 minutes at 5000 x g followed by germ-free filtration of the supernatant over 0.2 μηι PES filters (Nalgene).

Example 12: Protein concentration determination with TCA-biuret method

[00277] Concentrated protein samples (supernatants) were diluted with water to a concentration between 2 and 8 mg/mL. Bovine serum albumin (BSA) dilutions (0, 1 , 2, 5, 8 and 10 mg/mL were made and included as samples to generate a calibration curve. 1 mL of each diluted protein sample was transferred into a 10-mL tube containing 1 mL of a 20% (w/v) trichloro acetic acid solution in water and mixed thoroughly. Subsequently, the tubes were incubated on ice water for one hour and centrifuged for 30 minutes, at 4°C and 6000 rpm. The supernatant was discarded and pellets were dried by inverting the tubes on a tissue and letting them stand for 30 minutes at room temperature. Next, 4-mL BioQuant Biuret reagent mix was added to the pellet in the tube and the pellet was solubilized upon mixing. Next, 1 mL water was added to the tube, the tube was mixed thoroughly and incubated at room temperature for 30 minutes. The absorption of the mixture was measured at 546 nm with a water sample used as a blank measurement and the protein concentration was calculated via the BSA calibration line.

Example 13: Protein Activity Assays

13.1 Determination of pH optima

[00278] pH optima are determined by first determining the range of enzyme concentration that reproducibly displays initial velocity kinetics at standard pH and temperature for the appropriate assay. Enzyme is then diluted to an amount within this range, divided into aliquots, and each aliquot is assayed simultaneously at the different pHs.

13.2 Protein activity-temperature profiles

[00279] Temperature optima are determined by first determining the range of enzyme concentration that reproducibly displays initial velocity kinetics at 40°C and at the enzyme's optimal pH (see Example 13.1) in the appropriate assay. Enzyme is then diluted to an amount within this range, divided into aliquots, and, where possible, each aliquot is assayed simultaneously at the different temperatures (e.g., when reaction is incubated in a dry bath heater, then transferred to a plate reader for endpoint measurement). Where simultaneous measurements at different temperatures are impossible (e.g., when reaction is incubated in a plate reader for continuous measurement) activities are measured in sequence at different temperatures.

13.3 Assay Procedure CU1 : Colorimetric assay for qlvcosidase or esterase activity, measuring release of 4-nitrophenol [00280] Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L 10 μί of diluted enzyme sample is added to 30 pL of 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a PCR plate and preheated to appropriate temperature in a dry bath heater, and reaction is started by addition of 10 pL of preheated 5 mM substrate in water (see Table CU-1.1) to buffer and sample. Standards contain 10 pL of 4-nitrophenol (from 0 to 3 mM; 3 mM solution is made by dissolving 139 mg 4- nitrophenol in isopropyl alcohol and diluting 300 pL of resulting 100 mM solution to 10 mL in water) and 40 pL of reaction buffer. Sample blank contains 10 pL of enzyme sample and 40 pL of reaction buffer. Substrate blank contains 10 pL of substrate (see Table CU-1.1)) and 40 pL of reaction buffer. After appropriate incubation time, 50 pL of [1] for 4-nitrophenyl acetate, 1 M HEPES buffer pH 8 in water; [2] for all other substrates, 1 M Na ₂ C0 ₃ in water, is added. 80 pL is then transferred to a clear microtiter flat-bottomed plate, absorbance is read at 410 nm and compared to the standard curve. One unit is defined as the amount of enzyme that releases one micromole of 4-nitrophenol per minute at the specified pH and temperature. (Adapted from Holmsen et al (1989) Methods in Enzymology, 169, 336-342.)

Table CUM :

13.4 Assay Procedure CU2: Colorimetric assay for endo-qlvcanase activity, measuring copper (I) reduced by polysaccharide reducing ends

[00281] Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in water, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L. 10 pL of diluted sample is added to 30 pL of either [1] 50 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 3.42 mL 85% phosphoric acid, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) or [2] for enzymes that utilize calcium, 50 mM acetate-MOPS-borate reaction buffer at appropriate pH (made by dissolving 2.88 mL 99.7% glacial acetic acid, 10.45 g MOPS, and 3.10 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a PCR plate and preheated to appropriate temperature in a dry bath heater. The reaction is started by addition of 10 pL of preheated substrate in water (see Table CU-2.1) to buffer and sample. Standards contain 10 pL of 0 to 7.5 mM monosaccharide solution (see Table CU-2.1) in water and 40 pL of reaction buffer. Enzyme sample blank contains 10 pL of sample and 40 pL of reaction buffer. Substrate blank contains 10 pL of substrate (see Table CU-2.1) and 40 pL of reaction buffer. After appropriate incubation time, 10 pL is removed and added to another PCR plate containing 95 pL of BCA Reagent A (made by dissolving 0.543 g Na ₂ C0 ₃ , 0.242 g NaHCC>3 and 19 mg disodium 2,2'-bicinchoninate in water and diluting to 1 L) and 95 pL of BCA Reagent B (made by dissolving 12 mg CuS0 ₄ and 13 mg L-Serine in water and diluting to 1 L), sealed and incubated in a dry bath heater for 25 minutes at 80°C. PCR plate is put on ice for 5 minutes, then 160 pL is transferred to a clear microtiter flat-bottomed plate, absorbance is read at 562 nm and compared to the standard curve. One unit is defined as the amount of enzyme that releases one micromole of monosaccharide-equivalent reducing ends per minute at the specified pH and temperature. (Adapted from Fox et al (1991) Anal. Biochem., 195, 93-96). Colloidal chitin is prepared by mixing 10 g chitin from crab shell in 100 mL concentrated hydrochloric acid, stirring overnight at room temperature, then adding 1 L cold distilled water, filtering resulting suspension through Whatman No. 1 paper washing retentate with distilled water until pH is greater than 4, determining dry weight by gravimetry and diluting to 1 % solution with distilled water. (Adapted from Shimahara et al. (1988) Methods in Enzymology 161 , 417-423).

Table CU-2.1

Example 14: Identification of genes that encode secreted proteins

[00282] Genes (and polypeptides) from the organisms Amorphotheca resinae (Amore), Rhizomucor pusillus (Rhipu), and Calcarisporiella thermophila (Calth) were identified that, based on curation (described above, see Example 4), encoded a secreted protein. A list of these genes and polypeptides is shown in Tables 1A-1C.

Example 15: Characterization of expressed proteins

[00283] The Amorphotheca resinae proteins Amore2p4_006420, Amore2p4_001995, Amore2p4_010080, Amore2p4_002415, Amore2p4_008748, Amore2p4_002445, Amore2p4_006966, Amore2p4 006968, and Amore2p4_008748 were characterized using the assay protocols and assay conditions indicated in Table 5A. pH and temperature optima were determined for each protein as described in Examples 13.1 and 13.2. Results are shown in Table 5A.

[00284] The Rhizomucor pusillus proteins Rhipul p4_003290, Rhipul p4_002095, Rhipul p4_008391 , and Rhipu1 p4_009816 were characterized using the assay protocols and assay conditions indicated in Table 5B. pH and temperature optima were determined for each protein as described in Examples 13.1 and 13.2. Results are shown in Table 5B.

Table 5A: Activities of expressed enzymes from Amorphotheca resinae

* Control is an equal volume of supernatant from a vector-only transformant. na, not applicable as control exhibited no detectable activity.

* U, micromole product formed per minute under the indicated assay conditions

Table 5B: Activities of ex ressed enz mes from Rhizomucor usillus

* Control is an equal volume of supernatant from a vector-only transformant. na, not applicable as control exhibited no detectable activity.

* U, micromole product formed per minute under the indicated assay conditions

Example 16: Determination of activity-temperature profiles

[00285] Activity-temperature profiles were determined according to the protocol in Example 13.2 for the Amorphotheca resinae proteins Amore2p4_006420, Amore2p4_006966, Amore2p4_010080, Amore2p4_002445, Amore2p4_002415, Amore2p4_001995, Amore2p4_008748, and Amore2p4_006968, and the Rhizomucor pusillus proteins Rhipu1 p4_002095, Rhipu1p4_008391, Rhipu1p4_003290, and Rhipul p4_009816, using the Assay Protocols and Assay Conditions indicated below in Table 6. Results are shown in Figures 2-4.

Table 6: Activity-temperature profiles for various Amorphotheca resinae and Rhizomucor pusillus proteins

[00286] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.