NOVEL CELL WALL DECONSTRUCTION ENZYMES OF SCYTALIDIUM THERMOPHILUM, MYRIOCOCCUM THERMOPHILUM, AND AUREOBASIDIUM PULLULANS, AND USES THEREOF

Abstract
The present invention relates to novel polypeptides and enzymes (e.g., thermostable proteins 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, Scytalidium thermophilum strain CBS 625.91, Myriococcum thermophilum strain CBS 389.93, and Aureobasidium pullulans strain ATCC 62921. 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.
Description
FIELD OF THE INVENTION

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, Scytalidium thermophilum strain CBS 625.91, Myriococcum thermophilum strain CBS 389.93, and Aureobasidium pullulans strain ATCC 62921. 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

This application contains a Sequence Listing in computer readable form entitled “Seq_Listing_SCYTH_MYRTH_AURPU.txt”, created Jun. 6, 2013 having a size of about 7.78 MB. The computer readable form is incorporated herein by reference.


BACKGROUND OF THE INVENTION

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).


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.


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.


These enzymes may also be useful for other purposes in processing biomass. For example, the lignin modifying 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.


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.


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.


There is also a growing need for industrial 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).


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.


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

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, Scytalidium thermophilum strain CBS 625.91, Myriococcum thermophilum strain CBS 389.93, and Aureobasidium pullulans strain ATCC 62921, 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.


The soluble, secreted, biomass degradation proteins of the present invention comprise a proteome which is referred to herein as the SSBD proteome of Scytalidium thermophilum strain CBS 625.91, Myriococcum thermophilum, or Aureobasidium pullulans.


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: 571-855, 1468-1773, or 2548-2934
    • (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: 286-570, 1162-1467, or 2161-2547;
    • (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).


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


In some embodiments, the above mentioned polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 571-855, 1468-1773, or 2548-2934.


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


In some embodiments, above mentioned polypeptide is obtainable from a fungus. In some embodiments, the fungus is from the genus Scytalidium, Myriococcum, or Aureobasidium. In some embodiments, the fungus is Scytalidium thermophilum, Myriococcum thermophilum, or Aureobasidium pullulans.


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


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


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: 571-855, 1468-1773, or 2548-2934;
    • (b) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-285, 856-1161, or 1774-2160;
    • (c) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: SEQ ID NOs: 286-570, 1162-1467, or 2161-2547;
    • (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).


In some embodiments, the above mentioned polynucleotide molecule is obtainable from a fungus. In some embodiments, the fungus is from the genus Scytalidium, Myriococcum, or Aureobasidium. In some embodiments, the fungus is Scytalidium thermophilum, Myriococcum thermophilum, or Aureobasidium pullulans.


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.


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.


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 comprising a suitable carrier. In some embodiments, the composition further comprises a substrate of the polypeptide. In some embodiments, the substrate is biomass.


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.


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.


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.


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 embodiments, the food product is a bakery product.


In some aspects, the present invention relates to the use of any one of the above mentioned polypeptides for the preparation or processing of a food product. In some embodiments, the food product is a bakery 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 food product is a bakery product.


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.


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.


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.


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.


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


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.


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.


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.


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, N.Y.) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), Rieger et al., Glossary of genetics: Classical and molecular, 5th edition, Springer-Verlag, New-York, 1991; Alberts et al., Molecular Biology of the Cell, 4th 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).


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


DEFINITIONS

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.


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.


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 IUPAC-IUB Biochemical Nomenclature Commission.


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”.


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.


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”.


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).


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.


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 Intl 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′-O-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., 11th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Intl 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).


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.


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.


“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 Qβ-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. Pat. No. 6,087,133 and U.S. Pat. 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, 1173 1177; Lizardi et al., 1988, BioTechnology 6:1197 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.


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 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 1×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 5×SSC/5×Denhardt's solution/1.0% SDS and washing in 0.2×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).


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)×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.


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).


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): 444-453 (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 Blossom 62 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.


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, 4:11-17 (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.


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 NBLAST 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 NBLAST 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.qov/.


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 et al, Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).


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.


Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Pat. No. 5,503,980 (Cantor), U.S. Pat. No. 5,202,231 (Drmanac et al.), U.S. Pat. No. 5,149,625 (Church et al.), U.S. Pat. No. 5,112,736 (Caldwell et al.), U.S. Pat. No. 5,068,176 (Vijg et al.), and U.S. Pat. No. 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. Pat. Nos. 5,837,832 and 5,861,242 (Chee et al.)).


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.


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).


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).


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.


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.


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.


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.


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).


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.


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.


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.


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.


The phrase “cellulolytic enhancing activity” 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.


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 enhance 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

In the appended drawings:



FIG. 1 is a schematic map of the pGBFIN-49 expression plasmid.



FIG. 2 shows the endoxylanase activity of various secreted proteins from Scytalidium thermophilum (panel A), Myriococcum thermophilum (panel B), and Aureobasidium pullulans (panel C).



FIG. 3 shows the xyloglucanase activity of two secreted proteins from Aureobasidium pullulans on Tamarind xyloglucan.



FIGS. 4 and 5 show enzyme activity-temperature profiles of various secreted proteins from Scytalidium thermophilum.



FIGS. 6-11 show enzyme activity-temperature profiles of various secreted proteins from Myriococcum thermophilum.



FIGS. 12-16 show enzyme activity-temperature profiles of various secreted proteins from Aureobasidium pullulans.





In the appended Sequence Listing, SEQ ID NOs: 1-855 relate to sequences from Scytalidium thermophilum; SEQ ID NOs: 856-1773 relate to sequences from Myriococcum thermophilum; and SEQ ID NOs: 1774-2934 relate to sequences from Aureobasidium pullulans.


DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Polypeptides of the Invention

In one aspect, the present invention relates to isolated polypeptides secreted by Scytalidium thermophilum, Myriococcum thermophilum, or Aureobasidium pullulans, (e.g., Scytalidium thermophilum strain CBS 625.91, Myriococcum thermophilum strain CBS 389.93, or Aureobasidium pullulans strain ATCC 62921) having an activity relating to the processing or degradation of biomass (e.g., cell wall deconstruction).


In another aspect, the present invention relates to isolated polypeptides comprising the amino acid sequences shown in any one of SEQ ID NOs: 571-855, 1468-1773, or 2548-2934.


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.


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: 1-285, 856-1161, or 1774-2160), and coding (e.g., SEQ ID NOs: 286-570, 1162-1467, or 2161-2547) 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.


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: 1-285, 856-1161, or 1774-2160 (e.g., the intron or exon segments defined by the exon boundaries listed in Tables 2A-2C) or a functional part thereof.


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.


“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.


“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-(1-4) glucan consisting of anhydrocellobiose units. Endoglucanases, cellobiohydrolases, and beta-glucosidases are examples of cellulases.


“Endoglucanase” refers to a protein that catalyzes the hydrolysis of cellulose to oligosaccharide chains at random locations by means of an endoglucanase activity.


“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.


“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, mamians, 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.


“Xylanase” specifically refers to an enzyme that hydrolyzes the beta-1,4 bond in the xylan backbone, producing short xylooligosaccharides.


“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.


“Mannan endo-1,6-alpha-mannosidase” refers to a protein that hydrolyzes 1,6-alpha-mannosidic linkages in unbranched 1,6-mannans.


“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.


“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.


“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.


“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.


“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.


“Endo-arabinase” refers to a protein that catalyzes the hydrolysis of 1,5-alpha-arabinofuranosidic linkages in 1,5-arabinans.


“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.


“Beta-xylosidase” refers to a protein that hydrolyzes short 1,4-beta-D-xylooligomers into xylose.


“Cellobiose dehydrogenase” refers to a protein that oxidizes cellobiose to cellobionolactone.


“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).


“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.


“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.


“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).


“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.


“Lichenase” refers to a protein that catalyzes the hydrolysis of lichenan, a linear, 1,3-1,4-beta-D glucan.


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.


“Exo-rhamnogalacturonanase” refers to a protein that catalyzes the degradation of the rhamnogalacturonan backbone of pectin from the non-reducing end.


“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.


“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. Bacteria, 185:4727-4733 (2003)).


“Alpha-rhamnosidase” refers to a protein that catalyzes the hydrolysis of terminal non-reducing alpha-L-rhamnose residues in alpha-L-rhamnosides.


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.


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


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.


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) 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 1-4-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 (1-4)-alpha-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-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-arabi nofuranosidases; 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 1.11.1.13), which oxidizes lignin and lignin model compounds using Mn2+ and hydrogen peroxide; versatile peroxidases (EC 1.11.1.16), 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 1.11.1.13) 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.


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


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).


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).


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.


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.


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. 11:477).


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.


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 89:7811-7815; Delgrave et al., (1993) Protein Engineering 6(3): 327-331).


In another embodiment, functional variants of the present invention can encompasses 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.


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

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.


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

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.


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.


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

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.


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, Calif.). 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, N.J.).


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

The nucleic acid sequences of the genes disclosed herein were determined by sequencing cDNA clones, mRNA transcripts, or genomic DNA obtained from Scytalidium thermophilum strain CBS 625.9, Myriococcum thermophilum strain CBS 389.93, or Aureobasidium pullulans strain ATCC 62921.


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: 286-570, 1162-1467, or 2161-2547, or as set forth in Tables 1A-1C.


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: 1-285, 856-1161, or 1774-2160; or as set forth in Tables 1A-1C.


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: 1-285, 856-1161, or 1774-2160 (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.


In yet another aspect, the present invention relates to a polynucleotide comprising at least one exonic nucleic acid sequence comprised within SEQ ID NOs: 1-285, 856-1161, or 1774-2160 or as set forth in Tables 2A-2C.


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.


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.


As used herein, “very low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SDS at 45° C.


As used herein, “low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SDS at 50° C.


As used herein, “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SOS at 55° C.


As used herein, “medium-high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SDS at 60° C.


As used herein, “high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SDS at 65° C.


As used herein, “very high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×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 2×SSC, 0.2% SDS at 70° C.


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.


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.


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.


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.


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.


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, 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 to a polynucleotide of the present invention.


Sequencing Errors

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.


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.


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


Vectors

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


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.


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, Calif. (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).


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 T7 promoter regulatory sequences and T7 polymerase.


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.


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.


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, N.Y., 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.


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).


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.


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.


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.


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 by 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 by 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.


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

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.


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).


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.


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.


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).


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 organism. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S. Pat. No. 6,489,127. In 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 (CBS 389.93; ATCC 62921; or CBS 625.91).


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

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-1C, including to any fragment of any of the above sequences comprising at least one antibody binding epitope.


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.).


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.


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.


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

In another aspect, the present invention relates to 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).


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.


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

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.


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.


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.).


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.


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)). They have no known enzymatic activities of their own. 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 endogluconase, based on the measurement of very weak endo-1,4-β-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 to be classified in family 61 of the well-established CAZY GH classification system (http://www.cazy.org/GH61.html). The glycoside hydrolase family 61 is a member of the family of glycoside hydrolases EC 3.2.1. GH61 is used herein as being part of the cellulases.


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.


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.


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.


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


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.


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.


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

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.


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.


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 WO01/11974 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 NaCl 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.


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.


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.


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.


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).


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.


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

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

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.


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.


Non-starch polysaccharides (NSP) can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance.


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.


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.


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

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.


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.


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

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.


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.


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.


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.


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).


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

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.


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.


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.


In Tables 1A-1C below, the skilled person will recognize that although the precise protein activity of a polypeptide of the present invention may not be known (e.g., in the case of “unknown” proteins), the polypeptide may be nevertheless useful for carrying out an industrial process (e.g., cellulase-enhancing, cellulose-degrading, hemicellulose-degrading, etc.).









TABLE 1A







Biomass degrading genes and polypeptides of Scytalidium thermophilum
























Provisional
PCT


Gene ID in

Annotation in





application
application


Provisional

Provisional




CBM
SEQ ID NO:
SEQ ID NO:




















Application No.

Application No.



CAZy
of in-
Ge-
Cod-
Amino
Ge-
Cod-
Amino


61/657,082
Target ID
61/657,082
Updated annotation
Function
Protein activity
family
terest
nomic
ing
acid
nomic
ing
acid























Scyth2p4_000006
SCYTH_1_03938
xylanase
xylanase GH10
hemicellulose-
xylanase
GH10

1
2
3
1
286
571






degrading


Scyth2p4_000010
Scyth2p4_000010
unknown
Acid phosphatase
dephosphorylation
Acid phosphatase


4
5
6
2
287
572


Scyth2p4_000016

Leucine
Leucine
protein
protease


7
8
9
3
288
573




aminopeptidase 2
aminopeptidase 2
hydrolysis


Scyth2p4_000019
SCYTH_2_02709
unknown
unknown

uncharacterized


10
11
12
4
289
574







lignocellulose-induced







protein


Scyth2p4_000123

Putative beta-
arabinoxylan
hemicellulose-
arabinofuranosidase
GH43

13
14
15
5
290
575




xylosidase
arabinofuranohydrolase
degrading





GH43


Scyth2p4_000124
SCYTH_2_06066
unknown
unknown

uncharacterized


16
17
18
6
291
576







lignocellulose-induced







protein


Scyth2p4_000141

Probable aspartic-
Candidapepsin-3
protein
protease


19
20
21
7
292
577




type endopeptidase

hydrolysis




OPSB


Scyth2p4_000168

unknown
unknown

unknown


22
23
24
8
293
578


Scyth2p4_000230
Scyth2p4_000230
unknown
galactanase GH5
hemicellulose-
galactanase
GH5

25
26
27
9
294
579






degrading


Scyth2p4_000277

Putative lipase
Putative lipase
lipid-modifying
lipase


28
29
30
10
295
580




atg15
atg15


Scyth2p4_000610
Scyth2p4_000610
unknown
xylanase GH30
hemicellulose-
xylanase
GH30

31
32
33
11
296
581






degrading


Scyth2p4_000863
SCYTH_1_00740
hexosaminidase
hexosaminidase GH20
chitin-
hexosaminidase
GH20

34
35
36
12
297
582






degrading


Scyth2p4_000904
Scyth2p4_000904
Probable feruloyl
Probable feruloyl
hemicellulose-
feruloyl esterase


37
38
39
13
298
583




esterase A
esterase A
modifying


Scyth2p4_001035
Scyth2p4_001035
Tyrosinase
Tyrosinase
pigment-
Tyrosinase


40
41
42
14
299
584






generating


Scyth2p4_001183

Carboxypeptidase Y
Carboxypeptidase Y
protein
protease


43
44
45
15
300
585





homolog A
hydrolysis


Scyth2p4_001259
Scyth2p4_001259
unknown
unknown

uncharacterized


46
47
48
16
301
586







lignocellulose-induced







protein


Scyth2p4_001262
Scyth2p4_001262
endoglucanase
endoglucanase GH5
cellulose-
endoglucanase
GH5
CBM
49
50
51
17
302
587






degrading


1


Scyth2p4_001326

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


52
53
54
18
303
588






hydrolysis


Scyth2p4_001371
Scyth2p4_001371
Probable exo-1,4-
Beta-xylosidase GH3
hemicellulose-
beta-xylosidase
GH3

55
56
57
19
304
589




beta-xylosidase

degrading




bxlB


Scyth2p4_001379

Mannan endo-1,4-
beta-mannanase GH26
hemicellulose-
beta-mannanase
GH26
CBM
58
59
60
20
305
590




beta-mannosidase

degrading


35


Scyth2p4_001450

Carbohydrate-
possible carbohydrate-
carbohydrate-
carbohydrate-binding


61
62
63
21
306
591




binding cytochrome
binding cytochrome
oxidizing
cytochrome




b562 (Fragment)


Scyth2p4_001460
Scyth2p4_001460
chitinase
chitinase GH18
chitin-
chitinase
GH18

64
65
66
22
307
592






degrading


Scyth2p4_001513

Glucan 1,3-beta-
Glucan 1,3-beta-
cellulose-
glucan 1,3-beta-
GH55

67
68
69
23
308
593




glucosidase
glucosidase
degrading
glucosidase


Scyth2p4_001745
Scyth2p4_001745
Endoglucanase E1
Endoglucanase
cellulose-
endoglucanase
GH5

70
71
72
24
309
594






degrading


Scyth2p4_001867
SCYTH_1_00384
Probable beta-
Probable beta-
hemicellulose-
beta-mannosidase B
GH2

73
74
75
25
310
595




mannosidase B
mannosidase B
degrading


Scyth2p4_001875

Metallocarboxypeptidase
Metallocarboxypeptidase
protein
protease


76
77
78
26
311
596




A-like protein
A-like protein
hydrolysis




ARB_03789
MCYG_01475


Scyth2p4_001878
Scyth2p4_001878
unknown
unknown

uncharacterized


79
80
81
27
312
597







lignocellulose-induced







protein


Scyth2p4_001887
Scyth2p4_001887
O-
O-

oxidoreductase


82
83
84
28
313
598




methylsterigmatocystin
methylsterigmatocystin




oxidoreductase
oxidoreductase


Scyth2p4_001903

Probable leucine
Leucine
protein
protease


85
86
87
29
314
599




aminopeptidase
aminopeptidase 1
hydrolysis




MCYG_04170


Scyth2p4_001974

Endothiapepsin
Endothiapepsin
protein
protease


88
89
90
30
315
600






hydrolysis


Scyth2p4_001995
SCYTH_1_09959
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

91
92
93
31
316
601




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_001998
Scyth2p4_001998
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

94
95
96
32
317
602




esterase C

degrading


Scyth2p4_002014
Scyth2p4_002014
unknown
unknown

uncharacterized


97
98
99
33
318
603







lignocellulose-induced







protein


Scyth2p4_002032

unknown
unknown

unknown


100
101
102
34
319
604


Scyth2p4_002058

Tripeptidyl-
Tripeptidyl-
peptide
protease


103
104
105
35
320
605




peptidase sed1
peptidase sed1
hydrolysis


Scyth2p4_002089
SCYTH_1_01777
endo-1,5-alpha-
endo-1,5-alpha-
hemicellulose-
endo-1,5-alpha-
GH43

106
107
108
36
321
606




arabinanase
arabinanase GH43
degrading
arabinanase


Scyth2p4_002099

endoglucanase
Endoglucanase GH12
cellulose-
endoglucanase
GH12

109
110
111
37
322
607






degrading


Scyth2p4_002112

unknown
unknown

unknown CBM18

CBM
112
113
114
38
323
608









18


Scyth2p4_002143

Glucan 1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

115
116
117
39
324
609




glucosidase
glucanase GH55
degrading
glucanase


Scyth2p4_002153
Scyth2p4_002153
Adhesin, putative
possible adhesin

adhesin


118
119
120
40
325
610


Scyth2p4_002186
SCYTH_2_02011
Rhamnogalacturonan
rhamnogalacturonan
pectin-
rhamnogalacturonan
CE12

121
122
123
41
326
611




acetylesterase
acetylesterase CE12
degrading
acetylesterase


Scyth2p4_002220
Scyth2p4_002220
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61
CBM
124
125
126
42
327
612




enhancing protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_002225
Scyth2p4_002225
Cellobiose
Cellobiose
lignin-
cellobiose


127
128
129
43
328
613




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Scyth2p4_002425

Uncharacterized
Uncharacterized

oxidoreductase


130
131
132
44
329
614




oxidoreductase
oxidoreductase




C30D10.05c
C30D10.05c


Scyth2p4_002446
Scyth2p4_002446
Adhesin protein
possible adhesin

adhesin


133
134
135
45
330
615




Mad1


Scyth2p4_002491

Adhesin protein
possible adhesin

adhesin
GH18

136
137
138
46
331
616




Mad1


Scyth2p4_002582

Adhesin protein
possible adhesin

adhesin


139
140
141
47
332
617




Mad1


Scyth2p4_002596

Subtilisin-like
Subtilisin-like
protein
protease


142
143
144
48
333
618




proteinase Spm1
proteinase Spm1
hydrolysis


Scyth2p4_002639
SCYTH_2_05416
unknown
unknown

uncharacterized


145
146
147
49
334
619







lignocellulose-induced







protein


Scyth2p4_002689
Scyth2p4_002689
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

148
149
150
50
335
620




enhancing protein
monooxygenase
degrading
monooxygenase


Scyth2p4_002854
SCYTH_1_03782
arabinogalactanase
arabinogalactanase GH53
hemicellulose-
arabinogalactanase
GH53

151
152
153
51
336
621






degrading


Scyth2p4_002859

Nucleotide exchange
Nucleotide exchange

nucleotide exchange


154
155
156
52
337
622




factor SIL1
factor SIL1

factor


Scyth2p4_003064
Scyth2p4_003064
alpha-amylase
Alpha-amylase GH13
starch-
alpha-amylase
GH13

157
158
159
53
338
623






degrading


Scyth2p4_003098
Scyth2p4_003098
Killer toxin subunits
Killer toxin subunits
chitin-
chitinase
GH18
CBM
160
161
162
54
339
624




alpha/beta
alpha/beta
degrading


18


Scyth2p4_003108

Probable beta-
Probable beta-
cellulose-
beta-glucosidase
GH17

163
164
165
55
340
625




glucosidase btgE
glucosidase btgE
degrading


Scyth2p4_003124

Probable endo-1,3(4)-
mixed-link glucanase
glucan-
mixed-link glucanase
GH16

166
167
168
56
341
626




beta-glucanase
GH16
degrading




AFUB_029980


Scyth2p4_003222
Scyth2p4_003222
Endoglucanase-5
endoglucanase GH45
cellulose-
endoglucanase
GH45

169
170
171
57
342
627






degrading


Scyth2p4_003248

Lysophospholipase
Lysophospholipase
phospholipid-
lipase


172
173
174
58
343
628






modifying


Scyth2p4_003738

Probable aspartic-
Probable aspartic-
protein
protease


175
176
177
59
344
629




type endopeptidase
type endopeptidase
hydrolysis




opsB
OPSB


Scyth2p4_003766
Scyth2p4_003766
unknown
unknown

unknown GH16
GH16

178
179
180
60
345
630


Scyth2p4_003836
Scyth2p4_003836
Cellobiose
Cellobiose
cellulose-
cellobiose

CBM
181
182
183
61
346
631




dehydrogenase
dehydrogenase
degrading
dehydrogenase

1


Scyth2p4_003875
SCYTH_1_01865
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
184
185
186
62
347
632




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_003882
SCYTH_1_09023
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

187
188
189
63
348
633






degrading


Scyth2p4_003909
Scyth2p4_003909
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

190
191
192
64
349
634




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_003923
Scyth2p4_003923
unknown
xylan alpha-1,2-
hemicellulose-
xylan alpha-1,2-
GH115

193
194
195
65
350
635





glucuronidase GH115
modifying
glucuronidase


Scyth2p4_003925

cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
196
197
198
66
351
636




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_003929

unknown
unknown

unknown


199
200
201
67
352
637


Scyth2p4_003943

exo-1,3-beta-
exo-1,3-beta-
glucan-
exo-1,3-beta-
GH55

202
203
204
68
353
638




glucanase
glucanase GH55
degrading
glucanase


Scyth2p4_004010

Endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10

205
206
207
69
354
639




xylanase

degrading


Scyth2p4_004018
Scyth2p4_004018
unknown
unknown

uncharacterized


208
209
210
70
355
640







lignocellulose-induced







protein


Scyth2p4_004025
Scyth2p4_004025
alpha-
arabinoxylan
hemicellulose-
arabinofuranosidase
GH62

211
212
213
71
356
641




arabinofuranosidase
arabinofuranohydrolase
degrading





GH62


Scyth2p4_004026
SCYTH_1_04528
Alpha-N-
Alpha-N-
hemicellulose-
arabinofuranosidase
GH43

214
215
216
72
357
642




arabinofuranosidase
arabinofuranosidase
degrading




2
2


Scyth2p4_004049
Scyth2p4_004049
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

217
218
219
73
358
643




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_004099
Scyth2p4_004099
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

220
221
222
74
359
644




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_004162
Scyth2p4_004162
Probable
Acetylxylan esterase 1
hemicellulose-
acetylxylan esterase
CE1

223
224
225
75
360
645




acetylxylan esterase
CE1
modifying




A


Scyth2p4_004197
Scyth2p4_004197
unknown
exo-1,3-beta-
galactan-
exo-1,3-beta-
GH43
CBM
226
227
228
76
361
646





galactanase GH43
degrading
galactanase

35


Scyth2p4_004205
SCYTH_1_00248
endoglucanase
endoglucanase GH6
cellulose-
endoglucanase
GH6
CBM
229
230
231
77
362
647






degrading


1


Scyth2p4_004235

Aspergillopepsin A
Aspartic protease PEP3
protein
protease


232
233
234
78
363
648






hydrolysis


Scyth2p4_004237
SCYTH_1_01221
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

235
236
237
79
364
649






degrading


Scyth2p4_004263

Non-Catalytic
xylanase GH10
Hemicellulose-
xylanase
GH10
CBM
238
239
240
80
365
650




module family

degrading


1




expansin


Scyth2p4_004293
SCYTH_1_08979
cellobiohydrolase
cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7
CBM
241
242
243
81
366
651






degrading


1


Scyth2p4_004317
Scyth2p4_004317
unknown
unknown

uncharacterized


244
245
246
82
367
652







lignocellulose-induced







protein


Scyth2p4_004650
Scyth2p4_004650
unknown
Uncharacterized protein

uncharacterized


247
248
249
83
368
653





SAOUHSC_02143

lignocellulose-induced







protein


Scyth2p4_004945
Scyth2p4_004945
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

250
251
252
84
369
654




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_004976

N-acyl-
phospholipase
phospholipid-
lipase


253
254
255
85
370
655




phosphatidylethanol

modifying




amine-hydrolyzing




phospholipase D


Scyth2p4_005037
Scyth2p4_005037
Pectate lyase plyB
Pectate lyase plyB
pectin-
pectate lyase
PL1

256
257
258
86
371
656






degrading


Scyth2p4_005092

unknown
unknown

unknown CE3
CE3

259
260
261
87
372
657


Scyth2p4_005093
Scyth2p4_005093
Cutinase
Cutinase CE5
cutin-degrading
cutinase
CE5

262
263
264
88
373
658


Scyth2p4_005094
Scyth2p4_005094
Cutinase
Cutinase CE5
cutin-degrading
cutinase
CE5

265
266
267
89
374
659


Scyth2p4_005146

Leucine
Leucine
protein
protease


268
269
270
90
375
660




aminopeptidase 1
aminopeptidase 1
hydrolysis


Scyth2p4_005307

unknown
unknown

unknown CBM18

CBM
271
272
273
91
376
661









18


Scyth2p4_005334
Scyth2p4_005334
Pectinesterase
pectin methylesterase
pectin-
pectinesterase
CE8

274
275
276
92
377
662





CE8
modifying


Scyth2p4_005335
Scyth2p4_005335
unknown
Beta-glucanase
glucan-
beta-glucanase
GH16

277
278
279
93
378
663






degrading


Scyth2p4_005384

unknown
unknown

unknown


280
281
282
94
379
664


Scyth2p4_005465
Scyth2p4_005465
unknown
unknown

unknown CE16
CE16

283
284
285
95
380
665


Scyth2p4_005588
Scyth2p4_005588
unknown
unknown

uncharacterized


286
287
288
96
381
666







lignocellulose-induced







protein


Scyth2p4_005596

unknown
unknown

unknown CE4
CE4

289
290
291
97
382
667


Scyth2p4_005646
SCYTH_2_00017
unknown
unknown

uncharacterized


292
293
294
98
383
668







lignocellulose-induced







protein


Scyth2p4_005692

Bifunctional
acetylxylan esterase CE4
hemicellulose-
acetylxylan esterase
CE4

295
296
297
99
384
669




xylanase/deacetylase

degrading


Scyth2p4_005696
Scyth2p4_005696
unknown
unknown

uncharacterized


298
299
300
100
385
670







lignocellulose-induced







protein


Scyth2p4_005712
SCYTH_2_07654
Probable 1,4-beta-
carbohydrate esterase
hemicellulose-
unknown CE16
CE16
CBM
301
302
303
101
386
671




D-glucan

modifying


1




cellobiohydrolase C


Scyth2p4_005714
SCYTH_2_02004
Acetylxylan
acetylxylan esterase CE1
hemicellulose-
acetylxylan esterase
CE1
CBM
304
305
306
102
387
672




esterase A

modifying


1


Scyth2p4_005722
Scyth2p4_005722
Aldose 1-epimerase
Aldose 1-epimerase

aldose epimerase


307
308
309
103
388
673


Scyth2p4_005760
SCYTH_1_00672
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

310
311
312
104
389
674




enhancing protein
monooxygenase
degrading
monooxygenase


Scyth2p4_005775
Scyth2p4_005775
Expansin-like
possible expansin
cellulase-
expansin


313
314
315
105
390
675




protein

enhancing


Scyth2p4_005777
Scyth2p4_005777
non-catalytic
possible expansin
cellulase-
expansin


316
317
318
106
391
676




module family

enhancing




expansin


Scyth2p4_005792
SCYTH_1_00771
alpha-glucuronidase
alpha-glucuronidase
hemicellulose-
alpha-glucuronidase
GH67

319
320
321
107
392
677





GH67
modifying
GH67


Scyth2p4_005865
SCYTH_1_09242
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
322
323
324
108
393
678




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_005894

Uncharacterized
Uncharacterized

unknown CE4
CE4

325
326
327
109
394
679




protein yjeA
protein yjeA


Scyth2p4_006005
Scyth2p4_006005
unknown
exo-arabinanase GH93
hemicellulose-
exo-arabinanase
GH93

328
329
330
110
395
680






degrading
GH93


Scyth2p4_006013
Scyth2p4_006013
Probable 1,4-beta-
Exoglucanase 1
cellulose-
Exoglucanase
GH7

331
332
333
111
396
681




D-glucan

degrading




cellobiohydrolase B


Scyth2p4_006014
Scyth2p4_006014
Beta-galactosidase
Beta-glucuronidase
hemicellulose-
Beta-glucuronidase
GH2

334
335
336
112
397
682






degrading


Scyth2p4_006016

cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
337
338
339
113
398
683




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_006040
Scyth2p4_006040
unknown
unknown

uncharacterized


340
341
342
114
399
684







lignocellulose-induced







protein


Scyth2p4_006061

Putative
Putative
protein
protease


343
344
345
115
400
685




metallocarboxypeptidase
metallocarboxypeptidase
hydrolysis




MCYG_04493
ecm14


Scyth2p4_006263

Lipase
Lipase
lipid-degrading
lipase
CE5

346
347
348
116
401
686


Scyth2p4_006265
Scyth2p4_006265
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase GH3
GH3

349
350
351
117
402
687






degrading


Scyth2p4_006499

Neutral alpha-
Glucosidase 2 subunit
glucoside-
glucosidase
GH31

352
353
354
118
403
688




glucosidase AB
alpha
degrading


Scyth2p4_006556

Probable
Probable glycosidase crf1
glucoside-
glycosidase
GH16

355
356
357
119
404
689




glycosidase crf1

degrading


Scyth2p4_006566
SCYTH_2_05810
unknown
unknown

uncharacterized
GH43

358
359
360
120
405
690







lignocellulose-induced







protein


Scyth2p4_006586

cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

361
362
363
121
406
691




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_006591
Scyth2p4_006591
endoglucanase
xyloglucanase GH74
xyloglucan-
xyloglucanase
GH74
CBM
364
365
366
122
407
692






degrading


1


Scyth2p4_006628
Scyth2p4_006628
Carbohydrate-
possible carbohydrate-
carbohydrate-
carbohydrate-binding


367
368
369
123
408
693




binding cytochrome
binding cytochrome
oxidizing
cytochrome




b562


Scyth2p4_006768

exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

370
371
372
124
409
694




glucanase
glucanase GH55
degrading
glucanase


Scyth2p4_006914
SCYTH_2_07965
Putative
rhamnogalacturonan lyase
pectin-
rhamnogalacturonase
PL4

373
374
375
125
410
695




rhamnogalacturonase
PL4
degrading


Scyth2p4_006916
Scyth2p4_006916
Carbohydrate-
Cellobiose dehydrogenase
cellulose-
carbohydrate-binding


376
377
378
126
411
696




binding cytochrome

degrading
cytochrome




b562 (Fragment)


Scyth2p4_006920
SCYTH_2_04020
Exoglucanase-6A
possible expansin
cellulase-
expansin
CE3
CBM
379
380
381
127
412
697






enhancing


1


Scyth2p4_006931

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


382
383
384
128
413
698






hydrolysis


Scyth2p4_006993
SCYTH_1_03721
cellobiohydrolase
cellobiohydrolase GH6
cellulose-
cellobiohydrolase1
GH6
CBM
385
386
387
129
414
699






degrading


1


Scyth2p4_007002
Scyth2p4_007002
Swollenin
possible swollenin
cellulase-
swollenin
CE15
CBM
388
389
390
130
415
700






enhancing


1


Scyth2p4_007064

Vacuolar protease A
Vacuolar protease A
protein
protease


391
392
393
131
416
701






hydrolysis


Scyth2p4_007097
SCYTH_1_03940
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
394
395
396
132
417
702




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_007200
Scyth2p4_007200
Carbohydrate-
possible carbohydrate-
carbohydrate-
carbohydrate-binding


397
398
399
133
418
703




binding cytochrome
binding cytochrome
oxidizing
cytochrome




b562


Scyth2p4_007231
Scyth2p4_007231
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

400
401
402
134
419
704




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_007246

unknown
unknown

unknown CBM18

CBM
403
404
405
135
420
705









18


Scyth2p4_007249
Scyth2p4_007249
unknown
unknown

unknown CE15
CE15

406
407
408
136
421
706


Scyth2p4_007259
Scyth2p4_007259
arabinoxylan
arabinoxylan
hemicellulose-
arabinofuranosidase
GH62

409
410
411
137
422
707




arabinofuranohydrolase
arabinofuranosidase
modifying





GH62


Scyth2p4_007263

Adhesin protein,
possible adhesin

adhesin


412
413
414
138
423
708




putative


Scyth2p4_007266
Scyth2p4_007266
unknown
xylan alpha-1,2-
hemicellulose-
xylan alpha-1,2-
GH115

415
416
417
139
424
709





glucuronidase GH115
modifying
glucuronidase


Scyth2p4_007287
Scyth2p4_007287
unknown
unknown

uncharacterized


418
419
420
140
425
710







lignocellulose-induced







protein


Scyth2p4_007304
Scyth2p4_007304
unknown
unknown

uncharacterized


421
422
423
141
426
711







lignocellulose-induced







protein


Scyth2p4_007313

Probable exo-1,4-
Beta-xylosidase GH3
hemicellulose-
beta-xylosidase
GH3

424
425
426
142
427
712




beta-xylosidase

degrading




bxlB


Scyth2p4_007314
Scyth2p4_007314
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

427
428
429
143
428
713




esterase C

degrading


Scyth2p4_007531

unknown
unknown

unknown CE2
CE2

430
431
432
144
429
714


Scyth2p4_007556
SCYTH_1_05275
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
433
434
435
145
430
715




protein
monooxygenase
degrading
monooxygenase

1


Scyth2p4_007557
Scyth2p4_007557
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

436
437
438
146
431
716




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_007647
Scyth2p4_007647
Probable beta-
Beta-galactosidase GH35
hemicellulose-
beta-galactosidase
GH35

439
440
441
147
432
717




galactosidase B

degrading


Scyth2p4_007651
SCYTH_1_05320
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

442
443
444
148
433
718




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_007699
Scyth2p4_007699
Endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10

445
446
447
149
434
719




xylanase

degrading


Scyth2p4_007856
Scyth2p4_007856
Endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10

448
449
450
150
435
720




xylanase

degrading


Scyth2p4_007921

Serine-type
Serine-type
protein
protease


451
452
453
151
436
721




carboxypeptidase F
carboxypeptidase F
hydrolysis


Scyth2p4_008285
Scyth2p4_008285
unknown
unknown

uncharacterized


454
455
456
152
437
722







lignocellulose-induced







protein


Scyth2p4_008294
Scyth2p4_008294
unknown
unknown
cellulase-
uncharacterized


457
458
459
153
438
723






enhancing
lignocellulose-







induced protein2


Scyth2p4_008312
Scyth2p4_008312
unknown
unknown

uncharacterized


460
461
462
154
439
724







lignocellulose-induced







protein


Scyth2p4_008328
SCYTH_1_09441
xylanase
xylanase GH11
hemicellulose-
xylanase3
GH11
CBM
463
464
465
155
440
725






degrading


1


Scyth2p4_008336

Cuticle-degrading
Proteinase R
protein
protease


466
467
468
156
441
726




protease

hydrolysis


Scyth2p4_008341
SCYTH_1_05851
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

469
470
471
157
442
727




enhancing protein
monooxygenase
degrading
monooxygenase


Scyth2p4_008344

unknown
unknown
cellulose-
unknown CBM1

CBM
472
473
474
158
443
728






binding


1


Scyth2p4_008363
SCYTH_1_00589
endoglucanase
endoglucanase GH6
cellulose-
endoglucanase
GH6

475
476
477
159
444
729






degrading


Scyth2p4_008372
SCYTH_1_01623
cellobiohydrolase
cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7

478
479
480
160
445
730






degrading


Scyth2p4_008392
Scyth2p4_008392
unknown
unknown

uncharacterized


481
482
483
161
446
731







lignocellulose-induced







protein


Scyth2p4_008399
Scyth2p4_008399
Cellobiose
Cellobiose
Icellulose-
cellobiose


484
485
486
162
447
732




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Scyth2p4_008411

Podosporapepsin
Podosporapepsin
protein
protease


487
488
489
163
448
733






hydrolysis


Scyth2p4_008417

cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

490
491
492
164
449
734




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_008418
Scyth2p4_008418
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

493
494
495
165
450
735




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_008663
Scyth2p4_008663
Putative
Putative

oxidoreductase


496
497
498
166
451
736




uncharacterized
uncharacterized




oxidoreductase
oxidoreductase




YDR541C
YDR541C


Scyth2p4_008755
Scyth2p4_008755
glucoamylase
glucoamylase GH15
starch-
glucoamylase
GH15

499
500
501
167
452
737






degrading


Scyth2p4_008830

Phospholipase D
Alkaline phosphatase D
phospholipid-
lipase


502
503
504
168
453
738






modifying


Scyth2p4_008896
SCYTH_1_01504
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

505
506
507
169
454
739




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_009014

Gamma-
Gamma-
peptide-
protease


508
509
510
170
455
740




glutamyltranspeptidase 2
glutamyltranspeptidase 1
modifying


Scyth2p4_009047
Scyth2p4_009047
Aldose 1-epimerase
Aldose 1-epimerase

aldose epimerase


511
512
513
171
456
741


Scyth2p4_009244

Aspartic-type
Aspartic-type
protein
protease


514
515
516
172
457
742




endopeptidase ctsD
endopeptidase ctsD
hydrolysis


Scyth2p4_009303
Scyth2p4_009303
Probable alpha-N-
alpha-arabinofuranosidase
hemicellulose-
arabinofuranosidase
GH51

517
518
519
173
458
743




arabinofuranosidase
GH51
degrading




A


Scyth2p4_009308
SCYTH_2_07268
Feruloyl esterase B
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

520
521
522
174
459
744






degrading


Scyth2p4_009393

Probable acetylxylan
Probable acetylxylan
hemicellulose-
acetylxylan esterase
CE1

523
524
525
175
460
745




esterase
esterase A
degrading


Scyth2p4_009418

Endothiapepsin
Aspartic protease pep1
protein
protease


526
527
528
176
461
746






hydrolysis


Scyth2p4_009426
Scyth2p4_009426
Probable acetylxylan
Probable acetylxylan
hemicellulose-
acetylxylan esterase
CE1

529
530
531
177
462
747




esterase A
esterase A
modifying


Scyth2p4_009442
Scyth2p4_009442
unknown
unknown

uncharacterized

CBM
532
533
534
178
463
748







lignocellulose-

1







induced protein


Scyth2p4_009463

Probable leucine
Leucine aminopeptidase 2
protein
protease


535
536
537
179
464
749




aminopeptidase 2

hydrolysis


Scyth2p4_009475

Tripeptidyl-
Tripeptidyl-peptidase sed2
peptide
protease


538
539
540
180
465
750




peptidase sed3

hydrolysis


Scyth2p4_009509
Scyth2p4_009509
beta-mannanase
Beta-mannanase GH5
hemicellulose-
beta-mannanase
GH5

541
542
543
181
466
751






degrading


Scyth2p4_009510
Scyth2p4_009510
Glucoamylase
Glucoamylase
starch-
glucoamylase
GH119

544
545
546
182
467
752






degrading


Scyth2p4_009516

unknown
unknown

unknown PL20
PL20

547
548
549
183
468
753


Scyth2p4_009525
Scyth2p4_009525
unknown
unknown

uncharacterized


550
551
552
184
469
754







lignocellulose-induced







protein


Scyth2p4_009550

Cuticle-degrading
Cuticle-degrading
protein
protease


553
554
555
185
470
755




protease
protease
hydrolysis


Scyth2p4_009554

unknown
unknown

unknown CE3
CE3

556
557
558
186
471
756


Scyth2p4_009565
SCYTH_1_00574
endoglucanase
endoglucanase GH7
cellulose-
endoglucanase
GH7

559
560
561
187
472
757






degrading


Scyth2p4_009569

Putative serine
Putative serine
protein
protease


562
563
564
188
473
758




protease K12H4.7
protease K12H4.7
hydrolysis


Scyth2p4_009610

unknown
Beta-glucanase
glucan-
beta-glucanase
GH16

565
566
567
189
474
759






degrading


Scyth2p4_009620
SCYTH_1_08974
endoglucanase
endoglucanase GH45
cellulose-
endoglucanase
GH45
CBM
568
569
570
190
475
760






degrading


1


Scyth2p4_009626
SCYTH_1_01831
arabinoxylan
arabinoxylan
hemicellulose-
arabinofuranosidase4
GH62
CBM
571
572
573
191
476
761




arabinofuranosidase
arabinofuranosidase
degrading


1





GH62


Scyth2p4_009629
Scyth2p4_009629
unknown
unknown

uncharacterized


574
575
576
192
477
762







lignocellulose-induced







protein


Scyth2p4_009651
Scyth2p4_009651
unknown
Endo-1,4-beta-
hemicellulose-
endo-1,4-beta-
CE1

577
578
579
193
478
763





xylanase Z
degrading
xylanase


Scyth2p4_009653
Scyth2p4_009653
xylanase
xylanase GH10
hemicellulose-
xylanase
GH10

580
581
582
194
479
764






degrading


Scyth2p4_009700
Scyth2p4_009700
cellobiohydrolase
cellobiohydrolase GH6
cellulose-
cellobiohydrolase
GH6

583
584
585
195
480
765






degrading


Scyth2p4_009707
Scyth2p4_009707
Cellobiose
Cellobiose
cellulose-
cellobiose


586
587
588
196
481
766




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Scyth2p4_009711

unknown
unknown

unknown


589
590
591
197
482
767


Scyth2p4_009720
Scyth2p4_009720
unknown
unknown

uncharacterized


592
593
594
198
483
768







lignocellulose-induced







protein


Scyth2p4_009765

Adhesin protein
possible adhesin

adhesin


595
596
597
199
484
769




Mad1


Scyth2p4_009796
SCYTH_1_00755
alpha-glucosidase
Alpha-glucosidase GH31
starch-
alpha-glucosidase
GH31

598
599
600
200
485
770






degrading


Scyth2p4_009823
Scyth2p4_009823
Expansin family
possible expansin
cellulase-
expansin


601
602
603
201
486
771




protein

enhancing


Scyth2p4_009929
Scyth2p4_009929
Chitinase 3
Chitinase-like protein
chitin-
chitinase
GH18

604
605
606
202
487
772





PB1E7.04c
degrading


Scyth2p4_010021
SCYTH_1_01020
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

607
608
609
203
488
773




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_010034

Adhesin protein
possible adhesin

adhesion


610
611
612
204
489
774




Mad1


Scyth2p4_010146

unknown
unknown

unknown CE1
CE1

613
614
615
205
490
775


Scyth2p4_010149
Scyth2p4_010149
Exoglucanase 1
Cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7

616
617
618
206
491
776






degrading


Scyth2p4_010269
Scyth2p4_010269
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1
CBM
619
620
621
207
492
777




esterase C

modifying


1


Scyth2p4_010278
Scyth2p4_010278
Endochitinase
Killer toxin subunits
chitin-
chitinase
GH18
CBM
622
623
624
208
493
778





alpha/beta
degrading


18


Scyth2p4_010280

unknown
unknown

unknown


625
626
627
209
494
779


Scyth2p4_010281

unknown
unknown

unknown


628
629
630
210
495
780


Scyth2p4_010291

probable aspartic-
probable aspartic-
protein
protease


631
632
633
211
496
781




type endopeptidase
type endopeptidase
hydrolysis




opsB
opsB


Scyth2p4_010295
Scyth2p4_010295
cutinase
cutinase CE5
cutin-degrading
cutinase
CE5

634
635
636
212
497
782


Scyth2p4_010361

choline
possible pyranose
sugar
pyranose


637
638
639
213
498
783




dehydrogenase
dehydrogenase
modifying
dehydrogenase


Scyth2p4_010373
Scyth2p4_010373
carbohydrate-
possible carbohydrate-
carbohydrate-
carbohydrate-binding


640
641
642
214
499
784




binding cytochrome
binding cytochrome
oxidizing
cytochrome




b562


Scyth2p4_010387

exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

643
644
645
215
500
785




glucanase
glucanase GH55
degrading
glucanase


Scyth2p4_010416

Choline
Choline

Choline


646
647
648
216
501
786




dehydrogenase
dehydrogenase

dehydrogenase


Scyth2p4_010423
Scyth2p4_010423
unknown
Uncharacterized

uncharacterized


649
650
651
217
502
787





protein YkgB

lignocellulose-induced







protein


Scyth2p4_010457
SCYTH_1_01114
xylanase
xylanase GH11
hemicellulose-
xylanase5
GH11

652
653
654
218
503
788






degrading


Scyth2p4_010462
Scyth2p4_010462
Probable endo-1,4-
Probable endo-1,4-
hemicellulose-
endo-1,4-beta-
GH11

655
656
657
219
504
789




beta-xylanase B
beta-xylanase B
degrading
xylanase B


Scyth2p4_010469

choline
possible pyranose
sugar-
pyranose


658
659
660
220
505
790




dehydrogenase
dehydrogenase
modifying
dehydrogenase


Scyth2p4_010519

Peptidase M20
Peptidase M20 domain-
protein
protease


661
662
663
221
506
791




domain-containing
containing protein
hydrolysis




protein C757.05c
SMAC_03666.2


Scyth2p4_010552

choline
possible pyranose
sugar-
pyranose


664
665
666
222
507
792




dehydrogenase
dehydrogenase
modifying
dehydrogenase


Scyth2p4_010553

chitinase A1
chitinase GH18
chitin-
chitinase
GH18

667
668
669
223
508
793






degrading


Scyth2p4_010743

cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

670
671
672
224
509
794




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_010756
Scyth2p4_010756
unknown
unknown

uncharacterized


673
674
675
225
510
795







lignocellulose-induced







protein


Scyth2p4_010779
SCYTH_1_01186
probable chitinase 3
acidic mammalian
chitin-
chitinase
GH18
CBM
676
677
678
226
511
796





chitinase
degrading


18


Scyth2p4_010780

unknown
unknown

unknown


679
680
681
227
512
797


Scyth2p4_010784

exo-beta-D-
exo-glucosaminidase GH2
chitin-
exo-glucosaminidase
GH2

682
683
684
228
513
798




glucosaminidase

degrading


Scyth2p4_010822
Scyth2p4_010822
Probable pectate
pectate lyase PL3
pectin-
pectate lyase
PL3

685
686
687
229
514
799




lyase D

degrading


Scyth2p4_010823

laminarinase
laminarinase GH55
glucan-
laminarinase
GH55

688
689
690
230
515
800






degrading


Scyth2p4_010825
Scyth2p4_010825
cellulase-GH5
galactanase GH5
hemicellulose-
galactanase
GH5

691
692
693
231
516
801






degrading


Scyth2p4_010857
Scyth2p4_010857
endo-1,4-beta-
xylanase GH11
hemicellulose-
xylanase
GH11

694
695
696
232
517
802




xylanase A

degrading


Scyth2p4_010865
SCYTH_1_04962
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

697
698
699
233
518
803




protein
monooxygenase
degrading
monooxygenase


Scyth2p4_010870

unknown
unknown

unknown


700
701
702
234
519
804


Scyth2p4_010884
Scyth2p4_010884
unknown
unknown

uncharacterized


703
704
705
235
520
805







lignocellulose-induced







protein


Scyth2p4_010894
Scyth2p4_010894
Acetylxylan esterase
acetylxylan esterase CE5
hemicellulose-
acetylxylan esterase
CE5
CBM
706
707
708
236
521
806






modifying


1


Scyth2p4_010898
SCYTH_1_00286
endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10
CBM
709
710
711
237
522
807




xylanase

degrading


1


Scyth2p4_010899

aminopeptidase Y
aminopeptidase Y
protein
protease


712
713
714
238
523
808






hydrolysis



Scyth2p4_001141

Probable glycosidase crf1
carbohydrate-
glycosidase
GH16




239
524
809






modifying



Scyth2p4_001257

unknown

uncharacterized





240
525
810







lignocellulose-induced







protein



Scyth2p4_001442

unknown

uncharacterized





241
526
811







lignocellulose-induced







protein



Scyth2p4_001768

trehalase
starch-
trehalase
GH37




242
527
812






degrading



Scyth2p4_002054

alpha-amylase GH13
starch-
alpha-amylase
GH13




243
528
813






degrading



Scyth2p4_003709

unknown

uncharacterized





244
529
814







lignocellulose-induced







protein



Scyth2p4_003954

unknown

uncharacterized

CBM



245
530
815







lignocellulose-induced

1







protein



Scyth2p4_004342

unknown

uncharacterized





246
531
816







lignocellulose-induced







protein



Scyth2p4_004817

unknown

uncharacterized





247
532
817







lignocellulose-induced







protein



Scyth2p4_005217

unknown

uncharacterized





248
533
818







lignocellulose-induced







protein



Scyth2p4_007345

tyrosinase
pigment-
tyrosinase





249
534
819






producing



Scyth2p4_007869

unknown

uncharacterized





250
535
820







lignocellulose-induced







protein



Scyth2p4_009477

unknown

uncharacterized





251
536
821







lignocellulose-induced







protein



Scyth2p4_009552

unknown

uncharacterized





252
537
822







lignocellulose-induced







protein



Scyth2p4_009704

Uncharacterized

uncharacterized





253
538
823





protein L662

lignocellulose-induced







protein



Scyth2p4_010302

unknown

uncharacterized





254
539
824







lignocellulose-induced







protein



Scyth2p4_010820

unknown

uncharacterized





255
540
825







lignocellulose-induced







protein



SCYTH_1_00385

Probable beta-
hemicellulose-
beta-mannosidase B
GH2




256
541
826





mannosidase B
degrading



SCYTH_1_00739

hexosaminidase GH20
chitin-
hexosaminidase
GH20




257
542
827






degrading


[Scyth2p4_006265]6
SCYTH_1_02579

beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3




258
543
828






degrading



SCYTH_1_03688

arabinoxylan
hemicellulose-
arabinoxylan
GH62




259
544
829





arabinofuranohydrolase
degrading
arabinofurano-





GH62

hydrolase



SCYTH_1_09019

xylanase GH10
hemicellulose-
xylanase7
GH10




260
545
830






degrading



SCYTH_2_05417

unknown

uncharacterized





261
546
831







lignocellulose-induced







protein



SCYTH_2_07393

Acetylxylan esterase 1
hemicellulose-
acetylxylan esterase 1
CE1
CBM



262
547
832





CE1
modifying


1



Scyth2p4_000071

Dipeptidyl peptidase 1
protein
protease





263
548
833





(Fragment)
hydrolysis



Scyth2p4_000786

unknown

unknown CE1
CE1




264
549
834



Scyth2p4_000879

unknown

unknown CE3
CE3




265
550
835



Scyth2p4_001265

Putative sterigmatocystin
lignin-
peroxidase





266
551
836





biosynthesis peroxidase
degrading





stcC



Scyth2p4_001349

Probable serine protease
protein
protease





267
552
837





EDA2
hydrolysis



Scyth2p4_002059

Aspartic proteinase
protein
protease





268
553
838





yapsin-3
hydrolysis



Scyth2p4_002062

Lipase 4
lipid-modifying
lipase
CE10




269
554
839



Scyth2p4_002618

possible pyranose
sugar-
pyranose





270
555
840





dehydrogenase
modifying
dehydrogenase



Scyth2p4_002885

possible adhesin

adhesin





271
556
841



Scyth2p4_003845

unknown

unknown CBM18

CBM



272
557
842









18



Scyth2p4_003921

Dipeptidyl peptidase 4
protein
protease
CE1




273
558
843






hydrolysis



Scyth2p4_003974

Lipase 2
lipid-modifying
lipase
CE10




274
559
844



Scyth2p4_003996

Minor extracellular
protein
protease





275
560
845





protease vpr
hydrolysis



Scyth2p4_004891

possible adhesin

adhesin





276
561
846



Scyth2p4_005785

Probable isoaspartyl
protein
protease





277
562
847





peptidase/L-asparaginase
hydrolysis





3



Scyth2p4_006840

Aspergillopepsin-2
protein
protease





278
563
848






hydrolysis



Scyth2p4_007340

Alcohol dehydrogenase
sugar-
pyranose





279
564
849





[acceptor]
modifiying
dehydrogenase



Scyth2p4_007698

Uncharacterized FAD-

oxidoreductase





280
565
850





linked oxidoreductase





yvdP



Scyth2p4_008300

Extracellular
protein
protease





281
566
851





metalloprotease
hydrolysis





Pa_2_14170



Scyth2p4_009549

Uncharacterized FAD-

oxidoreductase





282
567
852





linked oxidoreductase





yvdP



Scyth2p4_010449

Probable aspartic-type
protein
protease
GH109




283
568
853





endopeptidase
hydrolysis





AFUA_3G01220



Scyth2p4_010575

Subtilisin-like protease
protein
protease





284
569
854





CPC735_066880
hydrolysis



Scyth2p4_010881

Uncharacterized FAD-

oxidoreductase





285
570
855





linked oxidoreductase





yvdP






1For example, exoglucanase-6A




2Simiar to aromatic ring-cleavage diooxygenases, upregulated by organism upon growth on biomass




3For example, endo-1,4-beta-xylanase B




4For example, alpha-L-arabinofuranosidase axhA-2.




5For example, endo-1,4-beta-xylanase 1




6Square brackets (“[” and “]”) used in this column in Tables 1A-1C are meant to indicate the possibility that the Gene IDs may have been modified from the provisional application.




7For example, Endo-1,4-beta-xylanase














TABLE 1B







Biomass degrading genes and polypeptides of Myriococcum thermophilum
























Provisional
PCT


Gene ID in

Annotation in





application
application


Provisional

Provisional




CBM
SEQ ID NO:
SEQ ID NO:




















Application No.

Application No.



CAZy
of in-
Ge-
Cod-
Amino
Ge-
Cod-
Amino


61/657,075
Target ID
61/657,075
Updated annotation
Function
Protein activity
family
terest
nomic
ing
acid
nomic
ing
acid























Myrth2p4_000015
Myrth2p4_000015
Putative beta-
arabinoxylan
hemicellulose-
arabinofuranosidase
GH43

1
2
3
856
1162
1468




xylosidase
arabinofuranohydrolase
degrading





GH43


Myrth2p4_000358
MYRTH_2_03236
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

4
5
6
857
1163
1469




enhancing protein
monooxygenase
degrading
monooxygenase


Myrth2p4_000359
Myrth2p4_000359
Cellobiose
Cellobiose
lignin-
cellobiose


7
8
9
858
1164
1470




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Myrth2p4_000363

Podosporapepsin
Podosporapepsin
protein
protease


10
11
12
859
1165
1471






hydrolysis


Myrth2p4_000376
Myrth2p4_000376
unknown
unknown

uncharacterized


13
14
15
860
1166
1472







lignocellulose-







induced protein


Myrth2p4_000388
MYRTH_2_00256
cellobiohydrolase
cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7

16
17
18
861
1167
1473






degrading


Myrth2p4_000417
Myrth2p4_000417
Acid phosphatase
Acid phosphatase
dephosphorylating
Acid phosphatase


19
20
21
862
1168
1474


Myrth2p4_000486

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


22
23
24
863
1169
1475






hydrolysis


Myrth2p4_000495
Myrth2p4_000495
unknown
arabinoxylan
hemicellulose-
arabinofuranosidase
GH43

25
26
27
864
1170
1476





arabinofuranohydrolase
degrading





GH43


Myrth2p4_000510
MYRTH_2_02564
unknown
unknown

uncharacterized


28
29
30
865
1171
1477







lignocellulose-







induced protein


Myrth2p4_000524

unknown
unknown

unknown


31
32
33
866
1172
1478


Myrth2p4_000531
Myrth2p4_000531
Uncharacterized
chitin deacetylase CE4
chitin-
chitin deacetylase
CE4

34
35
36
867
1173
1479




protein yjeA

degrading


Myrth2p4_000543

Probable aspartic-
Candidapepsin-8
protein
protease


37
38
39
868
1174
1480




type endopeptidase

hydrolysis




opsB


Myrth2p4_000545

unknown
unknown

unknown


40
41
42
869
1175
1481


Myrth2p4_000589

unknown
carbohydrate esterase
hemicellulose-
unknown CE15
CE15

43
44
45
870
1176
1482






modifying


Myrth2p4_000694

Putative lipase
Putative lipase
lipid-
lipase


46
47
48
871
1177
1483




atg15
atg15
degrading


Myrth2p4_000867

unknown
xylanase GH30
hemicellulose-
xylanase
GH30

49
50
51
872
1178
1484






degrading


Myrth2p4_000999
Myrth2p4_000999
unknown
unknown

uncharacterized


52
53
54
873
1179
1485







lignocellulose-







induced protein


Myrth2p4_001083

Carboxypeptidase Y
Carboxypeptidase Y
protein
protease


55
56
57
874
1180
1486





homolog A
hydrolysis


Myrth2p4_001208

Probable aspartic-
Probable aspartic-
protein
protease


58
59
60
875
1181
1487




type endopeptidase
type endopeptidase
hydrolysis




OPSB
OPSB


Myrth2p4_001304
Myrth2p4_001304
Cellobiose
Cellobiose
lignin-
cellobiose

CBM
61
62
63
876
1182
1488




dehydrogenase
dehydrogenase
degrading
dehydrogenase

1


Myrth2p4_001319

unknown
endo-beta-1,3-
hemicellulose-
endo-beta-1,3-
GH16

64
65
66
877
1183
1489





galactanase GH16
degrading
galactanase


Myrth2p4_001328
Myrth2p4_001328
unknown
unknown

uncharacterized


67
68
69
878
1184
1490







lignocellulose-







induced protein


Myrth2p4_001333
MYRTH_3_00119
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

70
71
72
879
1185
1491




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_001339
Myrth2p4_001339
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

73
74
75
880
1186
1492






degrading


Myrth2p4_001354
Myrth2p4_001354
endoglucanase
Probable xyloglucan-
hemicellulose-
xyloglucan-specific
GH12

76
77
78
881
1187
1493





specificendo-beta-1,4-
degrading
endo-beta-1,4-





glucanase A

glucanase A


Myrth2p4_001362
MYRTH_3_00118
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

79
80
81
882
1188
1494




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_001366
Myrth2p4_001366
unknown
unknown

uncharacterized


82
83
84
883
1189
1495







lignocellulose-







induced protein


Myrth2p4_001368

exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

85
86
87
884
1190
1496




glucanase
glucanase GH55
degrading
glucanase


Myrth2p4_001374
MYRTH_3_00100
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

88
89
90
885
1191
1497




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_001375
MYRTH_2_03396
xylanase
xylanase GH10
hemicellulose-
xylanase
GH10

91
92
93
886
1192
1498






degrading


Myrth2p4_001378
Myrth2p4_001378
unknown
unknown

uncharacterized


94
95
96
887
1193
1499







lignocellulose-







induced protein


Myrth2p4_001403
MYRTH_2_02621
Probable
Acetylxylan esterase 1
Hemicellulose-
acetylxylan esterase
CE1

97
98
99
888
1194
1500




acetylxylan esterase
CE1
modifying




A


Myrth2p4_001451
Myrth2p4_001451
xylanase
xylanase GH11
hemicellulose-
xylanase
GH11

100
101
102
889
1195
1501






degrading


Myrth2p4_001463
MYRTH_1_00071
chitinase
Chitinase GH18
chitin-
chitinase
GH18

103
104
105
890
1196
1502






degrading


Myrth2p4_001467

unknown
exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43
CBM
106
107
108
891
1197
1503





galactanase GH43
degrading
galactanase

35


Myrth2p4_001469

Tripeptidyl-
Tripeptidyl-
peptide
protease


109
110
111
892
1198
1504




peptidase sed2
peptidase sed3
hydrolysis


Myrth2p4_001494
Myrth2p4_001494
Probable beta-
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

112
113
114
893
1199
1505




glucosidase L

degrading


Myrth2p4_001496
Myrth2p4_001496
Probable exo-1,4-
beta-xylosidase GH3
hemicellulose-
beta-xylosidase8
GH3

115
116
117
894
1200
1506




beta-xylosidase

degrading




bxlB


Myrth2p4_001537
Myrth2p4_001537
Acetylxylan
Acetylxylan
hemicellulose-
acetylxylan
CE5

118
119
120
895
1201
1507




esterase 2
esterase 2 CE5
modifying
esterase


Myrth2p4_001550
Myrth2p4_001550
unknown
unknown

uncharacterized


121
122
123
896
1202
1508







lignocellulose-







induced protein


Myrth2p4_001581
MYRTH_2_03760
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

124
125
126
897
1203
1509




enhancing protein
monooxygenase
degrading
monooxygenase


Myrth2p4_001582
MYRTH_1_00083
Beta-galactosidase
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH2

127
128
129
898
1204
1510






degrading


Myrth2p4_001589

Putative serine
Probable serine
protein
protease


130
131
132
899
1205
1511




protease F56F10.1
protease EDA2
hydrolysis


Myrth2p4_001667

unknown
unknown

unknown CE4
CE4

133
134
135
900
1206
1512


Myrth2p4_001718
Myrth2p4_001718
unknown
unknown

unknown CE15
CE15

136
137
138
901
1207
1513


Myrth2p4_001719
MYRTH_2_02768
arabinogalactanase
arabinogalactanase
hemicellulose-
arabinogalactanase
GH53

139
140
141
902
1208
1514





GH53
degrading


Myrth2p4_001916
Myrth2p4_001916
Probable beta-
Probable beta-
cellulose-
beta-glucosidase
GH17

142
143
144
903
1209
1515




glucosidase btgE
glucosidase btgE
degrading


Myrth2p4_001926

Probable endo-1,3(4)-
mixed-link glucanase
glucan-
mixed-link
GH16

145
146
147
904
1210
1516




beta-glucanase
GH16
degrading
glucanase




NFIA_089530


Myrth2p4_001996
Myrth2p4_001996
endoglucanase
endoglucanase GH45
cellulose-
endoglucanase
GH45

148
149
150
905
1211
1517






degrading


Myrth2p4_002010

Lysophospholipase
Lysophospholipase
lipid-
lipase


151
152
153
906
1212
1518






modifying


Myrth2p4_002134

Putative
Aspartic protease pep1
protein
protease


154
155
156
907
1213
1519




aspergillopepsin A-

hydrolysis




like aspartic




endopeptidase




AFUA_2G15950


Myrth2p4_002293
Myrth2p4_002293
endoglucanase
Endoglucanase GH5
cellulose-
Endoglucanase
GH5
CBM
157
158
159
908
1214
1520






degrading


1


Myrth2p4_002328

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


160
161
162
909
1215
1521






hydrolysis


Myrth2p4_002394
MYRTH_2_04289
Mannanendo-1,4-
Beta-mannanase GH26
hemicellulose-
Beta-mannanase
GH26
CBM
163
164
165
910
1216
1522




beta-mannosidase

degrading


35


Myrth2p4_002434
MYRTH_1_00022
alpha-glucosidase
alpha-glucosidase GH31
starch-
alpha-glucosidase
GH31

166
167
168
911
1217
1523






degrading


Myrth2p4_002456

Carbohydrate-
unknown

Carbohydrate-


169
170
171
912
1218
1524




binding cytochrome


binding cytochrome




b562


Myrth2p4_002548

unknown
unknown

unknown


172
173
174
913
1219
1525


Myrth2p4_002549
Myrth2p4_002549
cellobiohydrolase
cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7

175
176
177
914
1220
1526






degrading


Myrth2p4_002563

Hybrid signal
Hybrid signal

kinase


178
179
180
915
1221
1527




transduction
transduction




histidine kinase J
histidine kinase J


Myrth2p4_002601
Myrth2p4_002601
Probable feruloyl
Probable feruloyl
hemicellulose-
feruloyl esterase


181
182
183
916
1222
1528




esterase A
esterase A
degrading


Myrth2p4_002632

unknown
Protoporphyrinogen

oxidoreductase


184
185
186
917
1223
1529





oxidase


Myrth2p4_002634
MYRTH_2_04579
hexosaminidase
Hexosaminidase GH20
chitin-
Hexosaminidase
GH20

187
188
189
918
1224
1530






degrading


Myrth2p4_002638
Myrth2p4_002638
Probable
Probable glycosidase
carbohydrate-
glycosidase
GH16

190
191
192
919
1225
1531




glycosidase CRH1
crf1
modifying


Myrth2p4_002915

Uncharacterized
Uncharacterized

oxidoreductase


193
194
195
920
1226
1532




oxidoreductase
oxidoreductase yusZ




C977.08/C1348.09


Myrth2p4_002916

Uncharacterized
Uncharacterized

oxidoreductase


196
197
198
921
1227
1533




oxidoreductase dltE
oxidoreductase dltE


Myrth2p4_002917

Glutaminyl-peptide
Glutaminyl-peptide
peptide-
Glutaminyl-peptide


199
200
201
922
1228
1534




cyclotransferase
cyclotransferase-like
modifying
cyclotransferase-





protein

like protein


Myrth2p4_002930
Myrth2p4_002930
beta-glucuronidase
beta-glucuronidase
hemicellulose-
beta-glucuronidase
GH79

202
203
204
923
1229
1535





GH79
degrading


Myrth2p4_003005
Myrth2p4_003005
Non-Catalytic
unknown

expansin


205
206
207
924
1230
1536




module family




expansin


Myrth2p4_003034
Myrth2p4_003034
unknown
Uncharacterized protein

Uncharacterized


208
209
210
925
1231
1537





YkgB

lignocellulose-







induced protein


Myrth2p4_003051
Myrth2p4_003051
unknown
unknown

uncharacterized


211
212
213
926
1232
1538







lignocellulose-







induced protein


Myrth2p4_003065
Myrth2p4_003065
unknown
unknown

uncharacterized


214
215
216
927
1233
1539







lignocellulose-







induced protein


Myrth2p4_003070
Myrth2p4_003070
exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

217
218
219
928
1234
1540




glucanase
glucanase GH55
degrading
glucanase


Myrth2p4_003103
MYRTH_3_00121
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

220
221
222
929
1235
1541




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_003203
Myrth2p4_003203
endoglucanase
cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7
CBM
223
224
225
930
1236
1542






degrading


1


Myrth2p4_003274
Myrth2p4_003274
Probable rhamno-
Probable rhamno-
pectin-
rhamno-


226
227
228
931
1237
1543




galacturonate
galacturonate
degrading
galacturonase




lyase C
lyase C


Myrth2p4_003333

unknown
exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43

229
230
231
932
1238
1544





galactanase GH43
degrading
galactanase


Myrth2p4_003368
MYRTH_1_00068
xylanase
Xylanase GH11
hemicellulose-
Xylanase
GH11

232
233
234
933
1239
1545






degrading


Myrth2p4_003495
Myrth2p4_003495
unknown
Uncharacterized protein

uncharacterized


235
236
237
934
1240
1546





SAOUHSC_02143

lignocellulose-







induced protein


Myrth2p4_003633
MYRTH_2_01655
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

238
239
240
935
1241
1547




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_003679

Lipase 1
Lipase 1
lipid-
lipase


241
242
243
936
1242
1548






degrading


Myrth2p4_003685
Myrth2p4_003685
unknown
xylanase GH30
hemicellulose-
xylanase
GH30

244
245
246
937
1243
1549






degrading


Myrth2p4_003686

N-acyl-
N-acyl-
phospholipid-
lipase


247
248
249
938
1244
1550




phosphatidylethanol-
phosphatidylethanol-
modifying




amine-hydrolyzing
amine-hydrolyzing




phospholipase D
phospholipase D


Myrth2p4_003747

unknown
unknown

unknown


250
251
252
939
1245
1551


Myrth2p4_003793

Probable leucine
Leucine aminopeptidase
protein
protease


253
254
255
940
1246
1552




aminopeptidase 1
1
hydrolysis


Myrth2p4_003921

unknown
unknown

unknown CBM18

CBM
256
257
258
941
1247
1553









18


Myrth2p4_003927
MYRTH_3_00104
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

259
260
261
942
1248
1554




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_003941
MYRTH_1_00024
unknown
Beta-glucanase
glucan-
Beta-glucanase
GH16

262
263
264
943
1249
1555






degrading


Myrth2p4_003942
Myrth2p4_003942
Pectinesterase A
pectin methylesterase
pectin-
pectinesterase
CE8

265
266
267
944
1250
1556





CE8
degrading


Myrth2p4_003966

unknown
unknown

unknown CBM18

CBM
268
269
270
945
1251
1557









18


Myrth2p4_004088

Lipase
Lipase
lipid-
lipase


271
272
273
946
1252
1558






degrading


Myrth2p4_004089
Myrth2p4_004089
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

274
275
276
947
1253
1559






degrading


Myrth2p4_004201

Putative
Putative
protein
protease


277
278
279
948
1254
1560




metallocarboxypeptidase
metallocarboxypeptidase
hydrolysis




MCYG_04493
ecm14


Myrth2p4_004260
MYRTH_2_04381
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61
CBM
280
281
282
949
1255
1561




protein
monooxygenase
degrading
monooxygenase

1


Myrth2p4_004335
MYRTH_2_03391
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

283
284
285
950
1256
1562




enhancing protein
monooxygenase
degrading
monooxygenase


Myrth2p4_004336
Myrth2p4_004336
endoglucanase
endoglucanase GH5
cellulose-
endoglucanase
GH5

286
287
288
951
1257
1563






degrading


Myrth2p4_004345

tripeptidyl-peptidase
tripeptidyl-peptidase
peptide
protease


289
290
291
952
1258
1564




sed2
sed2
hydrolysis


Myrth2p4_004391
MYRTH_201413
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61
CBM
292
293
294
953
1259
1565




enhancing protein
monooxygenase
degrading
monooxygenase

1


Myrth2p4_004393
Myrth2p4_004393
unknown
uncharacterized protein

uncharacterized


295
296
297
954
1260
1566





R656

lignocellulose-







induced protein


Myrth2p4_004397
MYRTH_300116
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

298
299
300
955
1261
1567




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_004415
MYRTH_100074
chitinase
Killer toxin subunits
chitin-
chitinase
GH18
CBM
301
302
303
956
1262
1568





alpha/beta
degrading


18


Myrth2p4_004442

Metallocarboxy-
Metallocarboxy-
protein
protease


304
305
306
957
1263
1569




peptidase A-like
peptidase A-like
hydrolysis




protein
protein




MCYG_01475
MCYG_01475


Myrth2p4_004455

galactanase
galactanase GH5
hemicellulose-
galactanase
GH30

307
308
309
958
1264
1570






degrading


Myrth2p4_004476
Myrth2p4_004476
unknown
unknown

uncharacterized


310
311
312
959
1265
1571







lignocellulose-







induced protein


Myrth2p4_004487
MYRTH_406966
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

313
314
315
960
1266
1572




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_004497
Myrth2p4_004497
Probable beta-
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH35

316
317
318
961
1267
1573




galactosidase B
GH35
degrading


Myrth2p4_004508
MYRTH_200518
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

319
320
321
962
1268
1574




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_004535
Myrth2p4_004535
Xylosidase/arabinosidase
Xylosidase/arabinosidase
Hemicellulose-
Xylosidase/arabinosidase
GH43

322
323
324
963
1269
1575






modifying


Myrth2p4_004704
Myrth2p4_004704
Putative galacturan
exo-
pectin-
rhamno-
GH28

325
326
327
964
1270
1576




1,4-alpha-
rhamnogalacturonase
degrading
galacturonase




galacturonidase B
GH28


Myrth2p4_004725

Carboxypeptidase
Carboxypeptidase
protein
protease


328
329
330
965
1271
1577




cpdS
cpdS
hydrolysis


Myrth2p4_004787

beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

331
332
333
966
1272
1578






degrading


Myrth2p4_004788
MYRTH_1_00011
Endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10

334
335
336
967
1273
1579




xylanase

degrading


Myrth2p4_004953
Myrth2p4_004953
unknown
unknown

uncharacterized


337
338
339
968
1274
1580







lignocellulose-







induced protein


Myrth2p4_004960
Myrth2p4_004960
unknown
unknown

uncharacterized


340
341
342
969
1275
1581







lignocellulose-







induced protein


Myrth2p4_004965
Myrth2p4_004965
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

343
344
345
970
1276
1582




esterase C

modifying


Myrth2p4_004966
Myrth2p4_004966
Feruloyl esterase B
feruloyl esterase CE1
Hemicellulose-
feruloyl esterase
CE1

346
347
348
971
1277
1583






modifyin


Myrth2p4_004986
MYRTH_2_01976
xylanase
xylanase GH11
hemicellulose-
xylanase
GH11
CBM
349
350
351
972
1278
1584






degrading


1


Myrth2p4_004993

Cuticle-degrading
Cuticle-degrading
protein
protease


352
353
354
973
1279
1585




protease
protease
hydrolysis


Myrth2p4_005017
MYRTH_3_00097
endoglucanase
endoglucanase GH6
cellulose-
endoglucanase
GH6

355
356
357
974
1280
1586






degrading


Myrth2p4_005025

Glucan 1,3-beta-
Glucan 1,3-beta-
cellulose-
Glucan 1,3-beta-
GH55

358
359
360
975
1281
1587




glucosidase
glucosidase
degrading
glucosidase


Myrth2p4_005037
Myrth2p4_005037
Carbohydrate-
Cellobiose
cellulose-
Carbohydrate-


361
362
363
976
1282
1588




binding cytochrome
dehydrogenase
degrading
binding cytochrome




b562 (Fragment)


Myrth2p4_005039

unknown
unknown

unknown CE15
CE15

364
365
366
977
1283
1589


Myrth2p4_005084
MYRTH_1_00077
xylanase
xylanase GH11
hemicellulose-
xylanase
GH11

367
368
369
978
1284
1590






degrading


Myrth2p4_005133
Myrth2p4_005133
unknown
unknown

uncharacterized


370
371
372
979
1285
1591







lignocellulose-







induced protein


Myrth2p4_005148
MYRTH_2_01934
unknown
carbohydrate esterase
hemicellulose-
unknown CE16
CE16

373
374
375
980
1286
1592






modifying


Myrth2p4_005149
Myrth2p4_005149
Acetylxylan
acetylxylan esterase CE1
hemicellulose-
acetylxylan esterase
CE1

376
377
378
981
1287
1593




esterase A

modifying


Myrth2p4_005155
Myrth2p4_005155
Aldose 1-epimerase
Aldose 1-epimerase

Aldose epimerase


379
380
381
982
1288
1594


Myrth2p4_005177
Myrth2p4_005177
unknown
unknown

uncharacterized


382
383
384
983
1289
1595







lignocellulose-







induced protein


Myrth2p4_005191
Myrth2p4_005191
Pectate lyase A
pectate lyase PL1
pectin-
pectate lyase
PL1

385
386
387
984
1290
1596






degrading


Myrth2p4_005222
MYRTH_2_03793
alpha-glucuronidase
alpha-glucuronidase
hemicellulose-
alpha-glucuronidase
GH67

388
389
390
985
1291
1597





GH67
modifyinging


Myrth2p4_005269
Myrth2p4_005269
unknown
unknown

uncharacterized


391
392
393
986
1292
1598







lignocellulose-







induced protein


Myrth2p4_005317
Myrth2p4_005317
unknown
xylan alpha-1,2-
hemicellulose-
xylan alpha-1,2-
GH11

394
395
396
987
1293
1599





glucuronidase GH115
modifying
glucuronidase
5







GH115


Myrth2p4_005320
MYRTH_2_00848
arabinoxylan
arabinoxylan
hemicellulose-
arabinofuranosidase
GH62

397
398
399
988
1294
1600




arabinofuranohydrolase
arabinofuranosidase
degrading





GH62


Myrth2p4_005321

Adhesin protein,
Major allergen Asp f 2

adhesin


400
401
402
989
1295
1601




putative


Myrth2p4_005328
Myrth2p4_005328
unknown
carbohydrate esterase
hemicellulose-
unknown CE15
CE15

403
404
405
990
1296
1602






modfiying


Myrth2p4_005329
Myrth2p4_005329
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

406
407
408
991
1297
1603




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_005340
Myrth2p4_005340
exo-
exo-
pectin-
exo-
GH28

409
410
411
992
1298
1604




polygalacturonase
polygalacturonase GH28
degrading
polygalacturonase


Myrth2p4_005343
MYRTH_2_04093
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

412
413
414
993
1299
1605




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_005368
Myrth2p4_005368
Carbohydrate-
unknown

Carbohydrate-


415
416
417
994
1300
1606




binding cytochrome


binding cytochrome




b562


Myrth2p4_005452
Myrth2p4_005452
beta-glucosidase
Beta-glucosidase GH3
cellulose-
Beta-glucosidase
GH3

418
419
420
995
1301
1607






degrading


Myrth2p4_005454
MYRTH_3_00103
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

421
422
423
996
1302
1608




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_005463

Carboxypeptidase
Carboxypeptidase
protein
protease


424
425
426
997
1303
1609




S1 homolog A
S1 homolog B
hydrolysis


Myrth2p4_005484

Vacuolar protease A
Vacuolar protease A
protein
protease


427
428
429
998
1304
1610






hydrolysis


Myrth2p4_005539
Myrth2p4_005539
Laccase-2
Laccase-2
lignin-
laccase


430
431
432
999
1305
1611






degrading


Myrth2p4_005561
Myrth2p4_005561
Probable feruloyl
Probable feruloyl
hemicellulose-
feruloyl esterase


433
434
435
1000
1306
1612




esterase B-1
esterase B-2
modifying


Myrth2p4_005590

Peptidase M20
Peptidase M20 domain-
protein
protease


436
437
438
1001
1307
1613




domain-containing
containing protein
hydrolysis




protein C757.05c
SMAC_03666.2


Myrth2p4_005626

Oryzin (protease)
Subtilisin-like protease 6
protein
protease


439
440
441
1002
1308
1614






hydrolysis


Myrth2p4_005639

chitinase
chitinase GH18
chitin-
chitinase
GH18

442
443
444
1003
1309
1615






degrading


Myrth2p4_005750
MYRTH_2_03494
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

445
446
447
1004
1310
1616




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_005752
MYRTH_2_01610
unknown
unknown

uncharacterized

CBM
448
449
450
1005
1311
1617







lignocellulose-

1







induced protein


Myrth2p4_005753
Myrth2p4_005753
unknown
unknown

uncharacterized


451
452
453
1006
1312
1618







lignocellulose-







induced protein


Myrth2p4_005819

laminarinase
Laminarinase GH55
Glucan-
Laminarinase
GH55

454
455
456
1007
1313
1619






degrading


Myrth2p4_005822
Myrth2p4_005822
unknown
unknown

uncharacterized


457
458
459
1008
1314
1620







lignocellulose-







induced protein


Myrth2p4_005854
MYRTH_1_00070
Probable endo-1,4-
Xylanase GH11
hemicellulose-
Xylanase
GH11

460
461
462
1009
1315
1621




beta-xylanase A

degrading


Myrth2p4_005856
MYRTH_1_00073
unknown
Beta-glucanase
glucan-
Beta-glucanase
GH16

463
464
465
1010
1316
1622






degrading


Myrth2p4_005886
Myrth2p4_005886
unknown
unknown

uncharacterized


466
467
468
1011
1317
1623







lignocellulose-







induced protein


Myrth2p4_005920

Leucine
Aminopeptidase Y
protein
protease


469
470
471
1012
1318
1624




aminopeptidase 2

hydrolysis


Myrth2p4_005923
Myrth2p4_005923
Acetylxylan esterase
acetylxylan esterase CE5
hemicellulose-
acetylxylan esterase
CE5
CBM
472
473
474
1013
1319
1625






degrading


1


Myrth2p4_005937

Aminopeptidase Y
Aminopeptidase Y
protein
protease


475
476
477
1014
1320
1626






hydrolysis


Myrth2p4_005945
MYRTH_1_00007
endo-1,5-alpha-
endo-1,5-alpha-
hemicellulose-
endo-1,5-alpha-
GH43

478
479
480
1015
1321
1627




arabinanase
arabinanase GH43
degrading
arabinanase


Myrth2p4_005946
Myrth2p4_005946
Alpha-N-
Alpha-N-
hemicellulose-
arabinofuranosidase
GH43

481
482
483
1016
1322
1628




arabinofuranosidase
arabinofuranosidase
degrading




2
2


Myrth2p4_005976
Myrth2p4_005976
endoglucanase
endoglucanase GH5
cellulose-
endoglucanase
GH5

484
485
486
1017
1323
1629






degrading


Myrth2p4_006001
Myrth2p4_006001
Laccase-1
Laccase-1
lignin-
laccase


487
488
489
1018
1324
1630






degrading


Myrth2p4_006022
Myrth2p4_006022
Probable pectin
pectin lyase PL1
pectin-
pectin lyase
PL1

490
491
492
1019
1325
1631




lyase A

degrading


Myrth2p4_006028
Myrth2p4_006028
unknown
galactanase GH5
hemicellulose-
galactanase
GH5

493
494
495
1020
1326
1632






degrading


Myrth2p4_006058

Bifunctional
chitin deacetylase CE4
chitin-
chitin deacetylase
CE4

496
497
498
1021
1327
1633




xylanase/deacetylase

modifying


Myrth2p4_006119
MYRTH_203560
endo-1,4-beta-
xylanase GH10
hemicellulose-
xylanase
GH10

499
500
501
1022
1328
1634




xylanase

degrading


Myrth2p4_006140
MYRTH_2_01176
alpha-arabino-
arabinoxylan arabino-
hemicellulose-
arabino-
GH62

502
503
504
1023
1329
1635




furanosidase
furanohydrolase GH62
degrading
furanosidase


Myrth2p4_006141
Myrth2p4_006141
Alpha-N-
Alpha-N-
hemicellulose-
arabinofuranosidase
GH43

505
506
507
1024
1330
1636




arabinofuranosidase
arabinofuranosidase
degrading




2
2


Myrth2p4_006201
Myrth2p4_006201
Cutinase
Cutinase CE5
cutin-
cutinase
CE5

508
509
510
1025
1331
1637






degrading


Myrth2p4_006226
Myrth2p4_006226
Probable pectate
pectate lyase PL1
pectin-
pectate lyase
PL1

511
512
513
1026
1332
1638




lyase B

degrading


Myrth2p4_006305
Myrth2p4_006305
cellobiohydrolase
Cellobiohydrolase GH7
cellulose-
Cellobiohydrolase
GH7

514
515
516
1027
1333
1639






degrading


Myrth2p4_006387

Probable
Probable
carbohydrate-
glycosidase
GH16

517
518
519
1028
1334
1640




glycosidase crf1
glycosidase crf1
modifying


Myrth2p4_006397
Myrth2p4_006397
beta-xylosidase
xylosidase/
hemicellulose-
xylosidase/
GH43

520
521
522
1029
1335
1641





arabinosidase
degrading
arabinosidase


Myrth2p4_006400
Myrth2p4_006400
unknown
unknown

uncharacterized
GH43

523
524
525
1030
1336
1642







lignocellulose-







induced protein


Myrth2p4_006403
MYRTH_2_04242
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

526
527
528
1031
1337
1643




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_006408
Myrth2p4_006408
endoglucanase
xyloglucanase GH74
hemicellulose-
xyloglucanase
GH74

529
530
531
1032
1338
1644






degrading
GH74


Myrth2p4_006434

Carbohydrate-
unknown
carbohydrate-
Carbohydrate-


532
533
534
1033
1339
1645




binding cytochrome

oxidizing
binding cytochrome




b562


Myrth2p4_006514

Subtilisin-like
Subtilisin-like
protein
protease


535
536
537
1034
1340
1646




proteinase Spm1
proteinase Spm1
hydrolysis


Myrth2p4_006524
Myrth2p4_006524
Adhesin protein
possible adhesin

adhesin


538
539
540
1035
1341
1647




Mad1


Myrth2p4_006587
MYRTH_1_00040
Chitinase 3
Endochitinase 2
chitin-
chitinase


541
542
543
1036
1342
1648






degrading


Myrth2p4_006646

Uncharacterized
Uncharacterized

oxidoreductase


544
545
546
1037
1343
1649




oxidoreductase
oxidoreductase




C30D10.05c
C30D10.05c


Myrth2p4_006765
Myrth2p4_006765
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

547
548
549
1038
1344
1650




esterase C

modifying


Myrth2p4_006772
Myrth2p4_006772
Cellobiose
Cellobiose
cellulose-
cellobiose


550
551
552
1039
1345
1651




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Myrth2p4_006795
MYRTH_2_04272
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61
CBM
553
554
555
1040
1346
1652




enhancing protein
monooxygenase
degrading
monooxygenase

1


Myrth2p4_006807
MYRTH_2_02340
Rhamnogalacturonan
rhamnogalacturonan
pectin-
rhamnogalacturonan
CE12

556
557
558
1041
1347
1653




acetylesterase
acetylesterase CE12
degrading
acetylesterase


Myrth2p4_006821
Myrth2p4_006821
Rhamnogalacturonan
Rhamnogalacturonan
pectin-
rhamnogalacturonan
CE12

559
560
561
1042
1348
1654




acetylesterase rhgT
acetylesterase rhgT
degrading
acetylesterase rhgT


Myrth2p4_006837
Myrth2p4_006837
Laccase-1
Laccase-1
lignin-
laccase


562
563
564
1043
1349
1655






degrading


Myrth2p4_007013

unknown
unknown

unknown


565
566
567
1044
1350
1656


Myrth2p4_007061
Myrth2p4_007061
Aldose 1-epimerase
Aldose 1-epimerase

Aldose epimerase


568
569
570
1045
1351
1657


Myrth2p4_007109

unknown
unknown

unknown


571
572
573
1046
1352
1658


Myrth2p4_007127

Beta-
Beta-
chitin-
Beta-
GH3

574
575
576
1047
1353
1659




hexosaminidase
hexosaminidase
degrading
hexosaminidase


Myrth2p4_007150
Myrth2p4_007150
Probable
Probable
hemicellulose-
acetylxylan esterase
CE1

577
578
579
1048
1354
1660




acetylxylan
acetylxylan
modifying




esterase A
esterase A


Myrth2p4_007367
MYRTH_2_02197
Feruloyl esterase B
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

580
581
582
1049
1355
1661






modifying


Myrth2p4_007409
Myrth2p4_007409
xylanase
xylanase GH11
hemicellulose-
xylanase
GH11

583
584
585
1050
1356
1662






degrading


Myrth2p4_007425
Myrth2p4_007425
unknown
unknown

uncharacterized

CBM
586
587
588
1051
1357
1663







lignocellulose-

1







induced protein


Myrth2p4_007444
Myrth2p4_007444
Cellobiose
Cellobiose
cellulose-
cellobiose


589
590
591
1052
1358
1664




dehydrogenase
dehydrogenase
degrading
dehydrogenase


Myrth2p4_007447

Carbohydrate-
Cellobiose
cellulose-
Carbohydrate-


592
593
594
1053
1359
1665




binding cytochrome
dehydrogenase
degrading
binding cytochrome




b562 (Fragment)


Myrth2p4_007461
MYRTH_3_00099
cellobiohydrolase
cellobiohydrolase GH6
cellulose-
cellobiohydrolase
GH6

595
596
597
1054
1360
1666






degrading


Myrth2p4_007538
MYRTH_2_00570
Putative rhamno-
rhamnogalacturonan
pectin-
rhamno-
PL4

598
599
600
1055
1361
1667




galacturonase
lyase PL4
degrading
galacturonate lyase


Myrth2p4_007539

Probable leucine
Leucine aminopeptidase
protein
protease


601
602
603
1056
1362
1668




aminopeptidase
1
hydrolysis




MCYG_04170


Myrth2p4_007540

Carbohydrate-
unknown
carbohydrate-
Carbohydrate-


604
605
606
1057
1363
1669




binding cytochrome

oxidizing
binding cytochrome




b562 (Fragment)


Myrth2p4_007556

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


607
608
609
1058
1364
1670






hydrolysis


Myrth2p4_007648

exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

610
611
612
1059
1365
1671




glucanase
glucanase GH55
degrading
glucanase


Myrth2p4_007688
Myrth2p4_007688
Manganese
Manganese
lignin-
manganese


613
614
615
1060
1366
1672




peroxidase 3
peroxidase 3
degrading
peroxidase


Myrth2p4_007726
Myrth2p4_007726
GLEYA adhesin
possible adhesin

adhesin


616
617
618
1061
1367
1673




domain


Myrth2p4_007729
MYRTH_1_00035
beta-mannanase
Beta-mannanase GH5
hemicellulose-
Beta-mannanase
GH5

619
620
621
1062
1368
1674






degrading


Myrth2p4_007771

Alpha-galactosidase
alpha-galactosidase
hemicellulose-
alpha-galactosidase
GH27

622
623
624
1063
1369
1675




A
GH27
degrading


Myrth2p4_007781

Probable leucine
Probable leucine
protein
protease


625
626
627
1064
1370
1676




aminopeptidase 2
aminopeptidase 2
hydrolysis


Myrth2p4_007801
Myrth2p4_007801
unknown
unknown

unknown


628
629
630
1065
1371
1677


Myrth2p4_007815
Myrth2p4_007815
xylanase
Xylanase GH10
hemicellulose-
Xylanase
GH10

631
632
633
1066
1372
1678






degrading


Myrth2p4_007838
Myrth2p4_007838
Pectate lyase H
pectate lyase PL3
pectin-
pectate lyase
PL3

634
635
636
1067
1373
1679






degrading


Myrth2p4_007849
Myrth2p4_007849
unknown
unknown

uncharacterized


637
638
639
1068
1374
1680







lignocellulose-







induced protein


Myrth2p4_007850
Myrth2p4_007850
unknown
unknown

uncharacterized


640
641
642
1069
1375
1681







lignocellulose-







induced protein


Myrth2p4_007861

Putative serine
Putative serine
protein
protease


643
644
645
1070
1376
1682




protease K12H4.7
protease K12H4.7
hydrolysis


Myrth2p4_007867
MYRTH_4_06111
endoglucanase
endoglucanase GH7
cellulose-
endoglucanase
GH7

646
647
648
1071
1377
1683






degrading


Myrth2p4_007877
MYRTH_2_00938
unknown
unknown

uncharacterized
CE3

649
650
651
1072
1378
1684







lignocellulose-







induced protein


Myrth2p4_007915
MYRTH_2_03335
unknown
Glucan endo-1,3-beta-
glucan-
Glucan endo-1,3-
GH16

652
653
654
1073
1379
1685





glucosidase A1
degrading
beta-glucosidase


Myrth2p4_007920

Subtilisin-like
Proteinase R
protein
protease


655
656
657
1074
1380
1686




protease 7

hydrolysis


Myrth2p4_007924
MYRTH_1_00018
Beta-glucuronidase
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH2

658
659
660
1075
1381
1687






degrading


Myrth2p4_007956

unknown
unknown

unknown PL20
PL20

661
662
663
1076
1382
1688


Myrth2p4_007996

Probable endo-
Probable endo-
glucan-
endo-1,3(4)-beta-
GH16

664
665
666
1077
1383
1689




1,3(4)-beta-
1,3(4)- beta-
degrading
glucanase




glucanase
glucanase




AFUB_029980
AFUA_2G14360


Myrth2p4_008028
MYRTH_2_00811
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

667
668
669
1078
1384
1690




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_008123
MYRTH_3_00077
unknown
unknown

unknown GH43
GH43

670
671
672
1079
1385
1691


Myrth2p4_008179
Myrth2p4_008179
unknown
unknown

unknown CE16
CE16

673
674
675
1080
1386
1692


Myrth2p4_008220
Myrth2p4_008220
unknown
unknown

uncharacterized


676
677
678
1081
1387
1693







lignocellulose-







induced protein


Myrth2p4_008285
Myrth2p4_008285
unknown
unknown

unknown CE4
CE4

679
680
681
1082
1388
1694


Myrth2p4_008298


endoglucanase GH12
cellulose-
endoglucanase
GH12

682
683
684
1083
1389
1695






degrading


Myrth2p4_008299
Myrth2p4_008299
Probable exo-1,4-
Beta-xylosidase GH3
hemicellulose-
Beta-xylosidase
GH3

685
686
687
1084
1390
1696




beta-xylosidase

degrading




xlnD


Myrth2p4_008353
Myrth2p4_008353
Periplasmic beta-
Periplasmic beta-
cellulose-
beta-glucosidase
GH3

688
689
690
1085
1391
1697




glucosidase
glucosidase
degrading


Myrth2p4_008360

Alpha-L-fucosidase
Alpha-L-fucosidase
carbohydrate-
Alpha-L-fucosidase
GH95

691
692
693
1086
1392
1698




2
2
modifying


Myrth2p4_008429

Putative
Putative

hydrolase


694
695
696
1087
1393
1699




uncharacterized
uncharacterized




hydrolase
hydrolase




YOR131C
YOR131C


Myrth2p4_008437
Myrth2p4_008437
Non-Catalytic
Allergen Asp f 7
cellulase-
expansin


697
698
699
1088
1394
1700




module family

enhancing




expansin


Myrth2p4_008501
Myrth2p4_008501
Carbohydrate-
unknown
carbohydrate-
Carbohydrate-


700
701
702
1089
1395
1701




binding cytochrome

oxidizing
binding cytochrome




b562


Myrth2p4_008515
MYRTH_4_03993
cellobiohydrolase
cellobiohydrolase GH6
cellulose-
cellobiohydrolase
GH6
CBM
703
704
705
1090
1396
1702






degrading


1


Myrth2p4_008522
Myrth2p4_008522
Probable 1,4-beta-
possible swollenin
cellulase-
swollenin
CE15
CBM
706
707
708
1091
1397
1703




D-glucan

enhancing


1




cellobiohydrolase C


Myrth2p4_008530
Myrth2p4_008530
cellulase-enhancing
polysaccharide
cellulose-
polysaccharide
GH61

709
710
711
1092
1398
1704




protein
monooxygenase
degrading
monooxygenase


Myrth2p4_008541

unknown
unknown

unknown CBM18

CBM
712
713
714
1093
1399
1705









18


Myrth2p4_008564
MYRTH_2_04212
unknown
unknown

uncharacterized


715
716
717
1094
1400
1706







lignocellulose-







induced protein


Myrth2p4_008615

exo-
exo-glucosaminidase
chitin-
exo-
GH2

718
719
720
1095
1401
1707




glucosaminidase
GH2
degrading
glucosaminidase


Myrth2p4_008650

unknown
unknown

unknown


721
722
723
1096
1402
1708


Myrth2p4_008756
Myrth2p4_008756
unknown
Endoglucanase
cellulose-
Endoglucanase
GH5

724
725
726
1097
1403
1709






degrading



Myrth2p4_000413

Cytochrome P450-DIT2

Cytochrome P450





1098
1404
1710



Myrth2p4_000624

unknown

uncharacterized





1099
1405
1711







lignocellulose-







induced protein



Myrth2p4_001189

Carboxylesterase 5A
carbohydrate-
carboxylesterase
CE10




1100
1406
1712






modifiying



Myrth2p4_001457

Cytochrome P450 52A12

Cytochrome P450





1101
1407
1713



Myrth2p4_001536

O-

oxidoreductase





1102
1408
1714





methylsterigmatocystin





oxidoreductase



Myrth2p4_001740

possible adhesin

adhesin





1103
1409
1715



Myrth2p4_003589

possible adhesin

adhesin





1104
1410
1716



Myrth2p4_003938

Tyrosinase
pigment-
Tyrosinase





1105
1411
1717






producing



Myrth2p4_006092

unknown

uncharacterized





1106
1412
1718







lignocellulose-







induced protein



Myrth2p4_006213

Tyrosinase
pigment-
Tyrosinase





1107
1413
1719






producing



Myrth2p4_008350

O-

oxidoreductase





1108
1414
1720





methylsterigmatocystin





oxidoreductase



MYRTH_1_00002

Alpha-L-arabino-
hemicellulose-
Alpha-L-arabino-
GH62




1109
1415
1721





furanosidase
degrading
furanosidase





(arabinoxylan

(arabinoxylan





arabinofuranosidase)

arabino-





GH62

furanosidase)


[Myrth2p4_008299]
MYRTH_1_00003

Probable exo-1,4-beta-
hemicellulose-
exo-1,4-beta-
GH3




1110
1416
1722





xylosidase xlnD
degrading
xylosidase



MYRTH_1_00009

exo-polygalacturonase
pectin-
exo-
GH28




1111
1417
1723





GH28
degrading
polygalacturonase



MYRTH_1_00020

exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43




1112
1418
1724





galactanase GH43
degrading
galactanase


[Myrth2p4_001494]
MYRTH_1_00021

Probable beta-
cellulose-
beta-glucosidase
GH3




1113
1419
1725





glucosidase L
degrading



MYRTH_1_00025

Endo-1,4-beta-xylanase
hemicellulose-
Endo-1,4-beta-
GH10




1114
1420
1726





A
degrading
xylanase



MYRTH_1_00031

beta-galactosidase GH35
hemicellulose-
beta-galactosidase
GH35




1115
1421
1727






degrading



MYRTH_1_00032

beta-galactosidase GH35
hemicellulose-
beta-galactosidase
GH35




1116
1422
1728






degrading



MYRTH_1_00037

Alpha-N-
hemicellulose-
Alpha-N-
GH43




1117
1423
1729





arabinofuranosidase 2
degrading
arabinofuranosidase



MYRTH_1_00069

hexosaminidase GH20
chitin-
hexosaminidase
GH20




1118
1424
1730






degrading



MYRTH_1_00080

unknown

unknown CE3
CE3




1119
1425
1731



MYRTH_1_00084

xylanase GH30
hemicellulose-
xylanase
GH30




1120
1426
1732






degrading



MYRTH_1_00087

beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3




1121
1427
1733






degrading



MYRTH_1_00098

Probable glycosidase
carbohydrate-
glycosidase
GH16




1122
1428
1734





crf1
modifying



MYRTH_2_00218

endoglucanase GH12
cellulose-
endoglucanase
GH12




1123
1429
1735






degrading



MYRTH_2_00583

Chitinase 3
chitin-
Chitinase 3





1124
1430
1736






degrading



MYRTH_2_00740

unknown

unknown GH16
GH16




1125
1431
1737


[Myrth2p4_000495]
MYRTH_2_00959

arabinoxylan arabino-
hemicellulose-
arabinoxylan
GH43




1126
1432
1738





furanohydrolase GH43
degrading
arabinofurano-







hydrolase9



MYRTH_2_01076

Chitinase GH18
chitin-
Chitinase
GH18




1127
1433
1739






degrading



MYRTH_2_01077

Chitinase GH18
chitin-
Chitinase
GH18




1128
1434
1740






degrading



MYRTH_2_01097

cellobiohydrolase GH7
cellulose-
cellobiohydrolase
GH7




1129
1435
1741






degrading



MYRTH_2_01279

Beta-xylosidase GH3
hemicellulose-
Beta-xylosidase
GH3




1130
1436
1742






degrading



MYRTH_2_01280

Beta-xylosidase GH3
hemicellulose-
Beta-xylosidase
GH3




1131
1437
1743






degrading



MYRTH_2_02633

Periplasmic beta-
cellulose-
beta-glucosidase
GH3




1132
1438
1744





glucosidase
degrading



MYRTH_2_04091

xylanase GH10
hemicellulose-
xylanase
GH10
CBM



1133
1439
1745






degrading


1



MYRTH_2_04186

endoglucanase GH7
cellulose-
endoglucanase
GH7




1134
1440
1746






degrading



MYRTH_2_04244

endoglucanase GH6
cellulose-
endoglucanase
GH6




1135
1441
1747






degrading



MYRTH_2_04271

hexosaminidase GH20
chitin-
hexosaminidase
GH20




1136
1442
1748






degrading



MYRTH_2_04288

Mannan endo-1,4-beta-
hemicellulose-
Mannan endo-1,4-
GH26
CBM



1137
1443
1749





mannosidase
degrading
beta-mannosidase

35



MYRTH_3_00003

Beta-mannanase GH5
hemicellulose-
Beta-mannanase
GH5




1138
1444
1750






degrading



MYRTH_3_00016

unknown

unknown GH16
GH16




1139
1445
1751



MYRTH_3_00086

exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43




1140
1446
1752





galactanase GH43
degrading
galactanase



MYRTH_3_00105

Polysaccharide
cellulose-
Polysaccharide
GH61




1141
1447
1753





monooxygenase
degrading
monooxygenase



MYRTH_3_00120

Polysaccharide
cellulose-
Polysaccharide
GH61
CBM



1142
1448
1754





monooxygenase GH61
degrading
monooxygenase

1



MYRTH_3_00124

Polysaccharide
cellulose-
Polysaccharide
GH61




1143
1449
1755





monooxygenase GH61
degrading
monooxygenase



MYRTH_3_00127

Alpha-L-
hemicellulose-
Alpha-L-arabino-
GH62




1144
1450
1756





arabinofuranosidase C
degrading
furanosidase C





(arabinoxylan arabino-

(arabinoxylan





furanohydrolase) GH62

arabino-







furanohydrolase)



MYRTH_4_05758

arabinoxylan
hemicellulose-
arabinoxylan
GH62




1145
1451
1757





arabinofuranohydrolase
degrading
arabinofurano-





GH62

hydrolase



MYRTH_4_09372

xylanase GH10
hemicellulose-
xylanase
GH10
CBM



1146
1452
1758






degrading


1



MYRTH_4_09820

endoglucanase GH12
cellulose-
endoglucanase
GH12




1147
1453
1759






degrading



Myrth2p4_000387

possible pyranose
sugar-
pyranose





1148
1454
1760





dehydrogenase
modifying
dehydrogenase



Myrth2p4_000489

Lipase 1
lipid-
lipase
CE10




1149
1455
1761






degrading



Myrth2p4_001363

Probable dipeptidyl
protein
protease
CE10




1150
1456
1762





peptidase 4
hydrolysis



Myrth2p4_001546

possible pyranose
sugar-
pyranose





1151
1457
1763





dehydrogenase
modifying
dehydrogenase



Myrth2p4_002267

possible pyranose
sugar-
pyranose





1152
1458
1764





dehydrogenase
modifying
dehydrogenase



Myrth2p4_002365

Probable serine protease
protein
protease





1153
1459
1765





EDA2
hydrolysis



Myrth2p4_003086

possible pyranose
sugar-
pyranose





1154
1460
1766





dehydrogenase
modifying
dehydrogenase



Myrth2p4_004152

unknown

unknown GH61
GH61




1155
1461
1767



Myrth2p4_004330

unknown

unknown CE3
CE3




1156
1462
1768



Myrth2p4_004961

Extracellular
protein
protease





1157
1463
1769





metalloprotease
hydrolysis





Pa_2_14170



Myrth2p4_005807

Uncharacterized

oxidoreductase





1158
1464
1770





oxidoreductase dltE



Myrth2p4_005966

Lipase 4
lipid-
lipase
CE10




1159
1465
1771






degrading



Myrth2p4_006645

Putative oxidoreductase

oxidoreductase





1160
1466
1772





C1F5.03c



Myrth2p4_008594

Uncharacterized FAD-

oxidoreductase





1161
1467
1773





linked oxidoreductase





yvdP






8For example, xylan 1,4-beta-xylosidase




9A minor activity of xylan 1,4-beta-xylosidase was detected for this protein.














TABLE 1C







Biomass degrading genes and polypeptides of Aureobasidium pullulans
























Provisional



Gene ID in

Annotation in





application
PCT application


prov.

Provisional





SEQ ID NO:
SEQ ID NO:




















appn.

application No.



CAZy
CBM of
Ge-

Amino
Ge-

Amino


61/657,078
Target ID
61/657,078
Updated annotation
Function
Protein activity
family
interest
nomic
Coding
acid
nomic
Coding
acid























Aurpu2p4_000013
Aurpu2p4_000013
Beta-glucosidase
beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1

1
2
3
1774
2161
2548




14

degrading


Aurpu2p4_000017
Aurpu2p4_000017
Probable rhamno-
Endo-rhamno-
pectin-
rhamno-
GH28

4
5
6
1775
2162
2549




galacturonase A
galacturonase GH28
degrading
galacturonase


Aurpu2p4_000070
Aurpu2p4_000070
endoglucanase
Endoglucanase GH5
cellulose-
endoglucanase
GH5
CBM1
7
8
9
1776
2163
2550






degrading


Aurpu2p4_000074
AURPU_3_00185
beta-glucosidase
avenacinase GH3

avenacinase
GH3

10
11
12
1777
2164
2551


Aurpu2p4_000163
AURPU_3_00030
xyloglucanase
xyloglucanase GH12
hemicellulose-
xyloglucanase
GH12

13
14
15
1778
2165
2552






degrading


Aurpu2p4_000184

N-acyl-
phospholipase
phospholipid-
lipase


16
17
18
1779
2166
2553




phosphatidylethanolamine-

modifying




hydrolyzing




phospholipase D


Aurpu2p4_000224
Aurpu2p4_000224
Acetylxylan
Acetylxylan esterase 1
hemicellulose-
acetylxylan
CE1

19
20
21
1780
2167
2554




esterase A
CE1
degrading
esterase


Aurpu2p4_000225
Aurpu2p4_000225
Putative Expansin-
Expansin-B5
cellulase-
expansin


22
23
24
1781
2168
2555




like protein 1

enhancing


Aurpu2p4_000232
Aurpu2p4_000232
unknown
unknown

unknown CE1
CE1

25
26
27
1782
2169
2556


Aurpu2p4_000354

unknown
unknown

unknown GH79
GH79

28
29
30
1783
2170
2557


Aurpu2p4_000408
Aurpu2p4_000408
Putative cell wall
possible adhesin

adhesin


31
32
33
1784
2171
2558




adhesin


Aurpu2p4_000459
Aurpu2p4_000459
exo-1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH5

34
35
36
1785
2172
2559




glucanase
glucanase GH5
degrading
glucanase


Aurpu2p4_000533

exo-1,3-beta-
Exo-1,3-beta-
cellulose-
Exo-1,3-beta-
GH55

37
38
39
1786
2173
2560




glucanase
glucanase GH55
degrading
glucanase


Aurpu2p4_000568
AURPU_3_00012
xylanase
xylanase GH10
hemicellulose-
xylanase
GH10
CBM1
40
41
42
1787
2174
2561






degrading


Aurpu2p4_000586

unknown
unknown

unknown


43
44
45
1788
2175
2562


Aurpu2p4_000590
AURPU_3_00002
Beta-glucosidase
beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1

46
47
48
1789
2176
2563




40

degrading


Aurpu2p4_000594

alpha-
alpha-galactosidase
hemicellulose-
alpha-
GH27

49
50
51
1790
2177
2564




galactosidase
GH27
degrading
galactosidase


Aurpu2p4_000617
Aurpu2p4_000617
Carboxylesterase 8
Para-nitrobenzyl

carboxylesterase
CE10

52
53
54
1791
2178
2565





esterase


Aurpu2p4_000662

Aspergillopepsin-F
Aspartic protease
protein
protease


55
56
57
1792
2179
2566





pep1
hydrolysis


Aurpu2p4_000692

beta-glucosidase
beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1

58
59
60
1793
2180
2567






degrading


Aurpu2p4_000730

Carboxypeptidase Y
Carboxypeptidase Y
protein
protease


61
62
63
1794
2181
2568





homolog A
hydrolysis


Aurpu2p4_000792
Aurpu2p4_000792
Probable pectin
pectin lyase PL1
pectin-degrading
pectin lyase
PL1

64
65
66
1795
2182
2569




lyase D


Aurpu2p4_000799

unknown
unknown

unknown CE1
CE1

67
68
69
1796
2183
2570


Aurpu2p4_000819

Putative serine
Putative serine
protein
protease


70
71
72
1797
2184
2571




protease K12H4.7
protease K12H4.7
hydrolysis


Aurpu2p4_000860
Aurpu2p4_000860
Probable alpha-N-
alpha
hemicellulose-
arabinofuranosidase
GH51

73
74
75
1798
2185
2572




arabinofuranosidase A
arabinofuranosidase
degrading





GH51


Aurpu2p4_000919
AURPU_3_00164
Putative
exo-
pectin-degrading
rhamno-
GH28

76
77
78
1799
2186
2573




galacturan 1,4-
rhamnogalacturonase

galacturonase




alpha-
GH28




galacturonidase B


Aurpu2p4_000934
AURPU_3_00165
Putative
exo-
pectin-degrading
rhamno-
GH28

79
80
81
1800
2187
2574




galacturan 1,4-
rhamnogalacturonase

galacturonase




alpha-
GH28




galacturonidase B


Aurpu2p4_000947
AURPU_3_00284
Inulinase
invertase GH32

invertase
GH32

82
83
84
1801
2188
2575


Aurpu2p4_000948
AURPU_3_00288
Invertase
exo-inulinase GH32

exo-inulinase
GH32

85
86
87
1802
2189
2576


Aurpu2p4_000984
AURPU_3_00187
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

88
89
90
1803
2190
2577






degrading


Aurpu2p4_000995
AURPU_3_00068
Probable endo-
mixed-link glucanase
glucan-
mixed-link
GH16

91
92
93
1804
2191
2578




1,3(4)-beta-
GH16
degrading
glucanase




glucanase




ACLA_073210


Aurpu2p4_001037
Aurpu2p4_001037
Cellobiose
Cellobiose
lignin-degrading
cellobiose


94
95
96
1805
2192
2579




dehydrogenase
dehydrogenase

dehydrogenase


Aurpu2p4_001097
Aurpu2p4_001097
Adhesin protein
possible adhesin

adhesin


97
98
99
1806
2193
2580




Mad1


Aurpu2p4_001104

unknown
galactanase GH5
hemicellulose-
galactanase
GH5

100
101
102
1807
2194
2581






degrading


Aurpu2p4_001152

Glucan 1,3-beta-
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

103
104
105
1808
2195
2582




glucosidase 1
beta-glucosidase A
degrading
glucosidase


Aurpu2p4_001194
Aurpu2p4_001194
Pectinesterase
Pectinesterase
pectin-degrading
pectinesterase
CE8

106
107
108
1809
2196
2583


Aurpu2p4_001195

Probable leucine
Leucine
protein
protease


109
110
111
1810
2197
2584




aminopeptidase 1
aminopeptidase 1
hydrolysis


Aurpu2p4_001256
AURPU_3_00017
xylanase
xylanase GH11
hemicellulose-
xylanase10
GH11

112
113
114
1811
2198
2585






degrading


Aurpu2p4_001441
Aurpu2p4_001441
Probable alpha-N-
alpha-
hemicellulose-
arabinofuranosidase
GH51

115
116
117
1812
2199
2586




arabinofuranosidase A
arabinofuranosidase
degrading





GH51


Aurpu2p4_001503
AURPU_3_00393
cellobiohydrolase
cellobiohydrolase GH6
cellulose-
cellobiohydrolase
GH6
CBM1
118
119
120
1813
2200
2587






degrading


Aurpu2p4_001504
AURPU_3_00429
cellobiohydrolase
cellobiohydrolase
cellulose-
cellobio-
GH7

121
122
123
1814
2201
2588





GH7
degrading
hydrolase11


Aurpu2p4_001512
Aurpu2p4_001512
Rhamnogalacturonase B
rhamnogalacturonan
pectin-degrading
rhamno-
PL4

124
125
126
1815
2202
2589





lyase PL4

galacturonase


Aurpu2p4_001553
Aurpu2p4_001553
Liver
Acetylcholinesterase 4

carboxylesterase
CE10

127
128
129
1816
2203
2590




carboxylesterase


Aurpu2p4_001599
Aurpu2p4_001599
Tannase
Tannase
tannin-
tannase


130
131
132
1817
2204
2591






degrading


Aurpu2p4_001600

Carboxypeptidase
Carboxypeptidase
protein
protease


133
134
135
1818
2205
2592




cpdS
cpdS
hydrolysis


Aurpu2p4_001633
Aurpu2p4_001633
endoglucanase
Endoglucanase GH5
cellulose-
Endoglucanase
GH5

136
137
138
1819
2206
2593






degrading


Aurpu2p4_001665

Gamma-
Gamma-
protein
protease


139
140
141
1820
2207
2594




glutamyltranspeptidase 2
glutamyltranspeptidase 1
hydrolysis


Aurpu2p4_001680

Peptidase M20
Probable
protein
protease


142
143
144
1821
2208
2595




domain-containing
carboxypeptidase
hydrolysis




protein C757.05c
AFLA_037450


Aurpu2p4_001713
Aurpu2p4_001713
Versatile
Manganese
lignin-degrading
versatile


145
146
147
1822
2209
2596




peroxidase VPL1
peroxidase 1

peroxidase


Aurpu2p4_001718
Aurpu2p4_001718
Endochitinase
chitinase GH18
chitin-degrading
chitinase
GH18

148
149
150
1823
2210
2597


Aurpu2p4_001807
AURPU_3_00342
unknown
Alpha-N-
hemicellulose-
arabino-
GH43

151
152
153
1824
2211
2598





arabinofuranosidase 2
degrading
furanosidase12


Aurpu2p4_001825
AURPU_3_00390
Probable
arabinogalactanase
hemicellulose-
arabino-
GH53

154
155
156
1825
2212
2599




arabinogalactan
GH53
degrading
galactanase




endo-1,4-beta-




galactosidase A


Aurpu2p4_001892
AURPU_3_00112
Probable
Probable glycosidase
carbohydrate-
glycosidase
GH16

157
158
159
1826
2213
2600




glycosidase CRH1
crf1
modifying


Aurpu2p4_001986
Aurpu2p4_001986
unknown
alpha-rhamnosidase
hemicellulose-
alpha-
GH78

160
161
162
1827
2214
2601





GH78
degrading
rhamnosidase


Aurpu2p4_002000

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


163
164
165
1828
2215
2602






hydrolysis


Aurpu2p4_002005

exo-arabinanase
exo-arabinanase
hemicellulose-
exo-arabinanase
GH93

166
167
168
1829
2216
2603





GH93
degrading


Aurpu2p4_002047
AURPU_3_00027
xylanase
xylanase GH11
hemicellulose-
xylanase
GH11

169
170
171
1830
2217
2604






degrading


Aurpu2p4_002086

Carboxypeptidase S
Carboxypeptidase S
protein
protease


172
173
174
1831
2218
2605






hydrolysis


Aurpu2p4_002155

unknown
unknown

unknown


175
176
177
1832
2219
2606


Aurpu2p4_002166
AURPU_3_00351
unknown
unknown

unknown
GH43

178
179
180
1833
2220
2607


Aurpu2p4_002167
Aurpu2p4_002167
Probable exo-1,4-
beta-xylosidase GH3
hemicellulose
beta-xylosidase
GH3

181
182
183
1834
2221
2608




beta-xylosidase

degrading




bxlB


Aurpu2p4_002190

Vacuolar protease A
Vacuolar protease A
protein
protease


184
185
186
1835
2222
2609






hydrolysis


Aurpu2p4_002220
Aurpu2p4_002220
Aldose 1-
Aldose 1-epimerase

aldose epimerase


187
188
189
1836
2223
2610




epimerase


Aurpu2p4_002256
AURPU_3_00237
Periplasmic beta-
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

190
191
192
1837
2224
2611




glucosidase

degrading


Aurpu2p4_002267
Aurpu2p4_002267
Acetylxylan
Acetylxylan esterase 2
hemicellulose-
acetylxylan
CE5

193
194
195
1838
2225
2612




esterase

degrading
esterase


Aurpu2p4_002284
Aurpu2p4_002284
alpha-arabino-
alpha-
hemicellulose-
arabino-
GH54

196
197
198
1839
2226
2613




furanosidase
arabinofuranosidase
degrading
furanosidase





GH54


Aurpu2p4_002399

unknown
unknown

unknown CBM18

CBM18
199
200
201
1840
2227
2614


Aurpu2p4_002518

Probable aspartic-
Probable aspartic-type
protein
protease


202
203
204
1841
2228
2615




type
endopeptidase OPSB
hydrolysis




endopeptidase




opsB


Aurpu2p4_002522
Aurpu2p4_002522
unknown
unknown

unknown GH43
GH43

205
206
207
1842
2229
2616


Aurpu2p4_002533
Aurpu2p4_002533
Laccase
Laccase-3 (Fragment)
lignin-degrading
laccase


208
209
210
1843
2230
2617


Aurpu2p4_002671
AURPU_3_00176
endo-
Endo-
pectin-degrading
Endo-
GH28

211
212
213
1844
2231
2618




polygalacturonase
polygalacturonase

polygalacturonase





GH28


Aurpu2p4_002672
AURPU_3_00239
beta-glucosidase
avenacinase GH3

avenacinase
GH3

214
215
216
1845
2232
2619


Aurpu2p4_002750

unknown
unknown

unknown CE16
CE16

217
218
219
1846
2233
2620


Aurpu2p4_002860
AURPU_3_00296
unknown
invertase GH32

invertase GH32
GH32

220
221
222
1847
2234
2621


Aurpu2p4_002907
AURPU_3_00353
unknown
unknown
cellulase -
unknown GH4313
GH43

223
224
225
1848
2235
2622






enhacing


Aurpu2p4_002940
Aurpu2p4_002940
Laccase-2
Laccase-1
lignin-degrading
laccase


226
227
228
1849
2236
2623


Aurpu2p4_002942

Aspergillopepsin-F
Aspartic protease
protein
protease


229
230
231
1850
2237
2624





pepB
hydrolysis


Aurpu2p4_002955

Putative
Putative
protein
protease


232
233
234
1851
2238
2625




metallocarboxypeptidase
metallocarboxypeptidase
hydrolysis




MCYG_04493
ECM14


Aurpu2p4_002987

unknown
unknown

unknown GH79
GH79

235
236
237
1852
2239
2626


Aurpu2p4_003029
Aurpu2p4_003029
Pectinesterase
pectin methylesterase
pectin-degrading
pectinesterase
CE8

238
239
240
1853
2240
2627





CE8


Aurpu2p4_003104

Probable glucan
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

241
242
243
1854
2241
2628




1,3-beta-
beta-glucosidase A
degrading
glucosidase




glucosidase D


Aurpu2p4_003184
AURPU_3_00468
Cellulase 1
Cellulase 1
cellulose-
cellulase
GH9

244
245
246
1855
2242
2629






degrading


Aurpu2p4_003313
Aurpu2p4_003313
Tannase
Tannase
Tannin-
tannase


247
248
249
1856
2243
2630






degrading


Aurpu2p4_003364
Aurpu2p4_003364
unknown
unknown

unknown CE1
CE1

250
251
252
1857
2244
2631


Aurpu2p4_003555

Lipase B
Lipase B
Lipid-degrading
lipase


253
254
255
1858
2245
2632


Aurpu2p4_003594
AURPU_3_00167
endo-
endo-
pectin-degrading
endo-
GH28

256
257
258
1859
2246
2633




polygalacturonase
polygalacturonase

polygalacturonase





GH28


Aurpu2p4_003606
Aurpu2p4_003606
Manganese
Manganese
lignin-degrading
manganese


259
260
261
1860
2247
2634




peroxidase 3
peroxidase 1

peroxidase


Aurpu2p4_003607
Aurpu2p4_003607
Versatile
Ligninase LG5
lignin-degrading
versatile


262
263
264
1861
2248
2635




peroxidase VPL1


peroxidase


Aurpu2p4_003685

unknown
unknown

unknown


265
266
267
1862
2249
2636


Aurpu2p4_003727

Putative
Putative
protein
protease


268
269
270
1863
2250
2637




aspergillopepsin A-
aspergillopepsin A-like
hydrolysis




like aspartic
aspartic




endopeptidase
endopeptidase




AFUA_2G15950
AFUA_2G15950


Aurpu2p4_003747
AURPU_3_00306
beta-galactosidase
Beta-galactosidase
hemicellulose-
beta-galactosidase
GH35

271
272
273
1864
2251
2638





GH35
degrading


Aurpu2p4_003884
AURPU_3_00389
arabinogalactanase
arabinogalactanase
hemicellulose-
arabino-
GH53

274
275
276
1865
2252
2639





GH53
degrading
galactanase


Aurpu2p4_003888
Aurpu2p4_003888
Cutinase 3
Cutinase 2
cutin-degrading
cutinase
CE5

277
278
279
1866
2253
2640


Aurpu2p4_003893
Aurpu2p4_003893
Expansin family
unknown

expansin


280
281
282
1867
2254
2641




protein


Aurpu2p4_003941
Aurpu2p4_003941
unknown
unknown

unknown CE2
CE2

283
284
285
1868
2255
2642


Aurpu2p4_004107

Carboxypeptidase
Carboxypeptidase S1
protein
protease


286
287
288
1869
2256
2643




S1 homolog A
homolog A
hydrolysis


Aurpu2p4_004115
AURPU_3_00326
endo-1,5-alpha-
endo-1,5-alpha-
hemicellulose-
endo-1,5-alpha-
GH43

289
290
291
1870
2257
2644




arabinanase
arabinanase GH43
degrading
arabinanase


Aurpu2p4_004128
AURPU_3_00242
unknown
xylanase GH30
hemicellulose
xylanase
GH30

292
293
294
1871
2258
2645






degrading


Aurpu2p4_004186
Aurpu2p4_004186
unknown
unknown

unknown CE5
CE5

295
296
297
1872
2259
2646


Aurpu2p4_004265
AURPU_3_00191
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

298
299
300
1873
2260
2647






degrading


Aurpu2p4_004286

Glucan 1,3-beta-
Glucan 1,3-beta-
glucan-
glucan 1,3-beta-


301
302
303
1874
2261
2648




glucosidase
glucosidase I/II
degrading
glucosidase


Aurpu2p4_004297

GLEYA adhesin
possible adhesin

adhesin
GH16

304
305
306
1875
2262
2649




domain


Aurpu2p4_004347

Probable aspartic-
Probable aspartic-type
protein
protease


307
308
309
1876
2263
2650




type
endopeptidase opsB
hydrolysis




endopeptidase




opsB


Aurpu2p4_004477

Glucan 1,3-beta-
exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH55

310
311
312
1877
2264
2651




glucosidase
glucanase GH55
degrading
glucanase


Aurpu2p4_004489

Carboxypeptidase
Carboxypeptidase
protein
protease


313
314
315
1878
2265
2652




cpdS
cpdS
hydrolysis


Aurpu2p4_004524
Aurpu2p4_004524
Acetylxylan
unknown
hemicellulose-
acetylxylan
CE5

316
317
318
1879
2266
2653




esterase 2

degrading
esterase


Aurpu2p4_004527

Probable endo-
Probable endo-1,3(4)-
cellulose-
endo-1,3(4)-beta-
GH16

319
320
321
1880
2267
2654




1,3(4)-beta-
beta-glucanase
degrading
glucanase




glucanase
NFIA_089530




AFUB_029980


Aurpu2p4_004550
AURPU_3_00396
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

322
323
324
1881
2268
2655




enhancing protein
monooxygenase
degrading
monooxygenase


Aurpu2p4_004694
Aurpu2p4_004694
unknown
unknown

unknown CE16
CE16

325
326
327
1882
2269
2656


Aurpu2p4_004762

unknown
unknown

unknown


328
329
330
1883
2270
2657


Aurpu2p4_004776
Aurpu2p4_004776
Expansin-like
Expansin-yoaJ
cellulose-
expansin


331
332
333
1884
2271
2658




protein 5

enhancing


Aurpu2p4_004801

Carboxypeptidase Y
Carboxypeptidase S1
protein
protease


334
335
336
1885
2272
2659





homolog B
hydrolysis


Aurpu2p4_004899
AURPU_3_00009
beta-glucosidase
beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1

337
338
339
1886
2273
2660






degrading


Aurpu2p4_004916
Aurpu2p4_004916
GLEYA adhesin
possible adhesin

adhesin


340
341
342
1887
2274
2661




domain


Aurpu2p4_004926
AURPU_3_00324
Probable arabinan
endo-1,5-alpha-
hemicellulose-
endo-1,5-alpha-
GH43

343
344
345
1888
2275
2662




endo-1,5-alpha-L-
arabinanase GH43
degrading
arabinanase




arabinosidase B


Aurpu2p4_004937
Aurpu2p4_004937
Probable
rhamnogalacturonan
pectin-degrading
rhamno-
PL4

346
347
348
1889
2276
2663




rhamnogalacturonate
lyase PL4

galacturonase




lyase B


Aurpu2p4_004986
Aurpu2p4_004986
Probable glucan
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

349
350
351
1890
2277
2664




1,3-beta-
beta-glucosidase A
degrading
glucosidase A




glucosidase D


Aurpu2p4_005056
AURPU_3_00397
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

352
353
354
1891
2278
2665




enhancing protein
monooxygenase
degrading
monooxygenase


Aurpu2p4_005097
AURPU_3_00100
Probable
Probable glycosidase
Carbohydrate-
glycosidase
GH16

355
356
357
1892
2279
2666




glycosidase crf1
crf1
modifying


Aurpu2p4_005194
AURPU_3_00166
endo-
endo-
pectin-degrading
endo-
GH28

358
359
360
1893
2280
2667




polygalacturonase
polygalacturonase

polygalacturonase





GH28


Aurpu2p4_005236
AURPU_3_00290
exo-inulinase
exo-inulinase

exo-inulinase
GH32

361
362
363
1894
2281
2668





GH32/GH43

GH32/GH43


Aurpu2p4_005278
Aurpu2p4_005278
Bifunctional
chitin deacetylase CE4
chitin-degrading
chitin deacetylase
CE4

364
365
366
1895
2282
2669




xylanase/deacetylase


CE4


Aurpu2p4_005399
AURPU_3_00295
unknown
unknown

unknown GH43
GH43

367
368
369
1896
2283
2670


Aurpu2p4_005401
Aurpu2p4_005401
alpha-
alpha-
hemicellulose-
arabinofuranosidase
GH51

370
371
372
1897
2284
2671




arabinofuranosidase
arabinofuranosidase
degrading





GH51


Aurpu2p4_005519
AURPU_3_00354
unknown
unknown
Hemicellulose-
unknown GH43
GH43

373
374
375
1898
2285
2672






modifying


Aurpu2p4_005580
Aurpu2p4_005580
Probable glucan
Probable glucan 1,3-
glucan-
glucan 1,3-beta-
GH5

376
377
378
1899
2286
2673




1,3-beta-
beta-glucosidase A
degrading
glucosidase




glucosidase A


Aurpu2p4_005825
Aurpu2p4_005825
Probable endo-
mixed-link glucanase
Glucan-
mixed-link
GH16

379
380
381
1900
2287
2674




1,3(4)-beta-
GH16
degrading
glucanase




glucanase




An02g00850


Aurpu2p4_005865

Probable beta-
Probable glycosidase
Carbohydrate-
glycosidase
GH16

382
383
384
1901
2288
2675




fructosidase
crf1
modfying


Aurpu2p4_005914
AURPU_3_00184
Probable exo-1,4-
beta-xylosidase GH3
hemicellulose-
beta-xylosidase14
GH3

385
386
387
1902
2289
2676




beta-xylosidase

degrading




bxlB


Aurpu2p4_005929
Aurpu2p4_005929
Uncharacterized
Uncharacterized

oxidoreductase


388
389
390
1903
2290
2677




oxidoreductase
oxidoreductase




C521.03
C521.03


Aurpu2p4_006113
AURPU_3_00395
cellulase-
polysaccharide
Cellulose-
polysaccharide
GH61
CBM1
391
392
393
1904
2291
2678




enhancing
monooxygenase
degrading
monooxygenase




protein


Aurpu2p4_006128
AURPU_3_00058
unknown
unknown

unknown GH16
GH16

394
395
396
1905
2292
2679


Aurpu2p4_006160
AURPU_3_00320
Xylosidase/arabinosidase
Xylosidase/arabinosidase
Hemicellulose-
Xylosidase/
GH43

397
398
399
1906
2293
2680






modifying
arabinosidase


Aurpu2p4_006162

unknown
unknown

unknown GH79
GH79

400
401
402
1907
2294
2681


Aurpu2p4_006176

Probable alpha-
alpha-galactosidase
hemicellulose-
alpha-
GH27

403
404
405
1908
2295
2682




galactosidase A
GH27
degrading
galactosidase


Aurpu2p4_006179

Lipase 2
Lipase 1
Lipid-degrading
lipase


406
407
408
1909
2296
2683


Aurpu2p4_006195

Carboxypeptidase
Carboxypeptidase S1
protein
protease


409
410
411
1910
2297
2684




S1 homolog A
homolog A
hydrolysis


Aurpu2p4_006206

unknown
unknown

unknown CE1
CE1

412
413
414
1911
2298
2685


Aurpu2p4_006207
Aurpu2p4_006207
GLEYA adhesin
possible adhesin

adhesin


415
416
417
1912
2299
2686




domain


Aurpu2p4_006222
Aurpu2p4_006222
Tannase
Tannase
Tannin-
tannase


418
419
420
1913
2300
2687






degrading


Aurpu2p4_006237
Aurpu2p4_006237
Probable pectate
pectate lyase PL3
pectin-degrading
pectate lyase
PL3

421
422
423
1914
2301
2688




lyase F


Aurpu2p4_006246
AURPU_3_00192
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

424
425
426
1915
2302
2689






degrading


Aurpu2p4_006312
AURPU_3_00032
unknown
xyloglucanase GH12
hemicellulose-
xyloglucanase
GH12

427
428
429
1916
2303
2690






degrading


Aurpu2p4_006313
Aurpu2p4_006313
Probable
pectin methylesterase
pectin-degrading
pectinesterase
CE8

430
431
432
1917
2304
2691




pectinesterase A
CE8


Aurpu2p4_006392
AURPU_3_00331
unknown
unknown

unknown GH43
GH43

433
434
435
1918
2305
2692


Aurpu2p4_006557

Glucan 1,3-beta-
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

436
437
438
1919
2306
2693




glucosidase 1
beta-glucosidase A
degrading
glucosidase A


Aurpu2p4_006782
Aurpu2p4_006782
beta-glucosidase
beta-glucosidase
cellulose-
beta-glucosidase
GH3

439
440
441
1920
2307
2694





GH3
degrading


Aurpu2p4_006900
Aurpu2p4_006900
GLEYA adhesin
possible adhesin

adhesin


442
443
444
1921
2308
2695




domain


Aurpu2p4_006933

Tripeptidyl-
Tripeptidyl-peptidase
protein
protease


445
446
447
1922
2309
2696




peptidase sed1
SED1
hydrolysis


Aurpu2p4_007070
Aurpu2p4_007070
GLEYA adhesin
possible adhesin

adhesin


448
449
450
1923
2310
2697




domain


Aurpu2p4_007082
AURPU_3_00013
xylanase
xylanase GH10
hemicellulose-
xylanase15
GH10

451
452
453
1924
2311
2698






degrading


Aurpu2p4_007093
AURPU_3_00019
xylanase
xylanase GH11
hemicellulose-
xylanase16
GH11

454
455
456
1925
2312
2699






degrading


Aurpu2p4_007113

unknown
unknown PL22
Pectin-
unknown PL22
PL22

457
458
459
1926
2313
2700






degrading


Aurpu2p4_007124

unknown
unknown

unknown CBM1

CBM1
460
461
462
1927
2314
2701


Aurpu2p4_007126
AURPU_3_00356
unknown
unknown

unknown GH43
GH43

463
464
465
1928
2315
2702


Aurpu2p4_007149
Aurpu2p4_007149
unknown
unknown

unknown CBM1

CBM1
466
467
468
1929
2316
2703


Aurpu2p4_007160

Putative lipase
Putative lipase
Lipid-degrading
lipase


469
470
471
1930
2317
2704




ATG15-1
ATG15-1


Aurpu2p4_007177
AURPU_3_00035
Beta-galactosidase
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH42

472
473
474
1931
2318
2705






degrading


Aurpu2p4_007190
Aurpu2p4_007190
Endoglucanase B
Endoglucanase B
cellulose-
Endoglucanase B
GH5

475
476
477
1932
2319
2706






degrading


Aurpu2p4_007196
Aurpu2p4_007196
Probable
Probable
pectin-degrading
pectinesterase
CE8

478
479
480
1933
2320
2707




pectinesterase/pectinesterase
pectinesterase A




inhibitor 41


Aurpu2p4_007206
AURPU_3_00177
exo-
exo-polygalacturonase
pectin-degrading
exo-
GH28

481
482
483
1934
2321
2708




polygalacturonase
GH28

polygalacturonase


Aurpu2p4_007220

Chitinase 1
Chitinase 3
chitin-degrading
chitinase
GH18

484
485
486
1935
2322
2709


Aurpu2p4_007270
AURPU_3_00241
beta-glucosidase
avenacinase GH3

avenacinase
GH3

487
488
489
1936
2323
2710


Aurpu2p4_007272

Carboxypeptidase
Carboxypeptidase
protein
protease


490
491
492
1937
2324
2711




cpdS
cpdS
hydrolysis


Aurpu2p4_007292

Putative
Putative NADPH-

oxidoreductase


493
494
495
1938
2325
2712




uncharacterized
dependent




oxidoreductase
methylglyoxal




YGL157W
reductase GRP2


Aurpu2p4_007342

unknown
unknown

unknown CE16
CE16

496
497
498
1939
2326
2713


Aurpu2p4_007356
AURPU_3_00178
exo-
exo-polygalacturonase
pectin-degrading
exo-
GH28

499
500
501
1940
2327
2714




polygalacturonase
GH28

polygalacturonase


Aurpu2p4_007383
Aurpu2p4_007383
unknown
unknown CE1
hemicellulose-
xylan alpha-1,2-
GH115

502
503
504
1941
2328
2715






degrading
glucuronidase


Aurpu2p4_007404

Probable glucan
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

505
506
507
1942
2329
2716




1,3-beta-
beta-glucosidase A
degrading
glucosidase




glucosidase A


Aurpu2p4_007424
Aurpu2p4_007424
unknown
galactanase GH5
hemicellulose-
galactanase
GH5

508
509
510
1943
2330
2717






degrading


Aurpu2p4_007428

unknown
exo-arabinanase
hemicellulose-
exo-arabinanase
GH93

511
512
513
1944
2331
2718





GH93
degrading


Aurpu2p4_007429
AURPU_3_00314
Alpha-N-
Alpha-N-
hemicellulose-
arabino-
GH43

514
515
516
1945
2332
2719




arabinofuranosidase 2
arabinofuranosidase 2
degrading
furanosidase


Aurpu2p4_007455
Aurpu2p4_007455
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase


517
518
519
1946
2333
2720




esterase B

degrading


Aurpu2p4_007488
AURPU_3_00028
unknown
Probable xyloglucan-
Hemicellulose-
xyloglucan-
GH12

520
521
522
1947
2334
2721





specific endo-beta-
degrading
specific endo-





1,4-glucanase A

beta-1,4-







glucanase


Aurpu2p4_007493

Subtilisin-like
Alkaline protease 2
protein
protease


523
524
525
1948
2335
2722




proteinase Spm1

hydrolysis


Aurpu2p4_007511
AURPU_3_00315
Alpha-N-
Alpha-N-
hemicellulose-
arabino-
GH43

526
527
528
1949
2336
2723




arabinofuranosidase 2
arabinofuranosidase 2
degrading
furanosidase


Aurpu2p4_007612
Aurpu2p4_007612
Cutinase
cutinase CE5
cutin-degrading
cutinase
CE5

529
530
531
1950
2337
2724


Aurpu2p4_007614
Aurpu2p4_007614
Probable pectin
pectin lyase PL1
pectin-degrading
pectin lyase
PL1

532
533
534
1951
2338
2725




lyase A


Aurpu2p4_007621
AURPU_3_00155
endo-
Endo-
pectin-degrading
Endo-
GH28

535
536
537
1952
2339
2726




polygalacturonase
polygalacturonase

polygalacturonase





GH28


Aurpu2p4_007662

Aspergillopepsin-2
Aspergillopepsin-2
protein
protease


538
539
540
1953
2340
2727






hydrolysis


Aurpu2p4_007707
AURPU_3_00394
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

541
542
543
1954
2341
2728




enhancing protein
monooxygenase
degrading
monooxygenase


Aurpu2p4_007805
Aurpu2p4_007805
Laccase-3
Laccase
lignin-degrading
laccase


544
545
546
1955
2342
2729




(Fragment)


Aurpu2p4_007919
Aurpu2p4_007919
unknown
unknown

unknown CE5
CE5

547
548
549
1956
2343
2730


Aurpu2p4_008001
AURPU_3_00054
unknown
unknown

unknown GH16
GH16

550
551
552
1957
2344
2731


Aurpu2p4_008021
Aurpu2p4_008021
Mannan endo-1,4-
Mannan endo-1,4-
hemicellulose-
Mannan endo-1,4-
GH5

553
554
555
1958
2345
2732




beta-mannosidase 3
beta-mannosidase 4
degrading
beta-mannosidase


Aurpu2p4_008140
Aurpu2p4_008140
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase


556
557
558
1959
2346
2733




esterase B-2

modifying


Aurpu2p4_008212
AURPU_3_00357
Putative
Putative
cellulose-
endoglucanase
GH45

559
560
561
1960
2347
2734




endoglucanase
endoglucanase type K
degrading




type K


Aurpu2p4_008231
AURPU_3_00157
Endo-
Probable endo-
pectin-degrading
endo-
CE8

562
563
564
1961
2348
2735




xylogalacturonan
xylogalacturonan

xylogalacturonan




hydrolase A
hydrolase A

hydrolase


Aurpu2p4_008239
AURPU_3_00323
unknown
exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43
CBM35
565
566
567
1962
2349
2736





galactanase GH43
degrading
galactanase


Aurpu2p4_008212
AURPU_3_00357
Putative
Putative
cellulose-
endoglucanase
GH45

559
560
561
1960
2347
2734




endoglucanase
endoglucanase type K
degrading




type K


Aurpu2p4_008231
AURPU_3_00157
Endo-
Probable endo-
pectin-degrading
endo-
CE8

562
563
564
1961
2348
2735




xylogalacturonan
xylogalacturonan

xylogalacturonan




hydrolase A
hydrolase A

hydrolase


Aurpu2p4_008239
AURPU_3_00323
unknown
exo-1,3-beta-
hemicellulose-
exo-1,3-beta-
GH43
CBM35
565
566
567
1962
2349
2736





galactanase GH43
degrading
galactanase


Aurpu2p4_008255
AURPU_3_00064
Beta-glucanase
unknown

unknown GH16
GH16

568
569
570
1963
2350
2737


Aurpu2p4_008271

Putative lipase
Putative lipase
lipid-degrading
lipase


571
572
573
1964
2351
2738




ATG15-1
ATG15-1


Aurpu2p4_008282

Probable
Probable tripeptidyl-
protein
protease


574
575
576
1965
2352
2739




tripeptidyl-
peptidase SED2
hydrolysis




peptidase SED2


Aurpu2p4_008385
Aurpu2p4_008385
Liver
Carboxylesterase 5A

carboxylesterase
CE10

577
578
579
1966
2353
2740




carboxylesterase


Aurpu2p4_008412
AURPU_3_00305
Probable beta-
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH35

580
581
582
1967
2354
2741




galactosidase C
GH35
degrading


Aurpu2p4_008485
AURPU_3_00101
Probable endo-
mixed-link glucanase
Glucan-
mixed-link
GH16

583
584
585
1968
2355
2742




1,3(4)-beta-
GH16
degrading
glucanase




glucanase




AFUB_029980


Aurpu2p4_008495
Aurpu2p4_008495
Unknown-Esterase
unknown

unknown CE12
CE12

586
587
588
1969
2356
2743


Aurpu2p4_008503
Aurpu2p4_008503
unknown
probable beta-
cellulase-
beta-
GH79

589
590
591
1970
2357
2744





glucuronidase GH79
enhacing
glucuronidase


Aurpu2p4_008585
Aurpu2p4_008585
Cellobiose
Cellobiose
lignin-degrading
cellobiose

CBM1
592
593
594
1971
2358
2745




dehydrogenase
dehydrogenase

dehydrogenase


Aurpu2p4_008692

Carboxypeptidase
Carboxypeptidase S1
protein
protease


595
596
597
1972
2359
2746




S1 homolog B
homolog A
hydrolysis


Aurpu2p4_008705
Aurpu2p4_008705
GLEYA adhesin
possible adhesin

adhesin


598
599
600
1973
2360
2747




domain


Aurpu2p4_008725
AURPU_3_00334
Arabinan endo-
endo-1,5-alpha-
hemicellulose-
endo-1,5-alpha-
GH43

601
602
603
1974
2361
2748




1,5-alpha-L-
arabinanase GH43
degrading
arabinanase




arabinosidase


Aurpu2p4_008775
Aurpu2p4_008775
Putative
Putative galacturan
pectin-degrading
galacturan 1,4-
GH28

604
605
606
1975
2362
2749




galacturan 1,4-
1,4-alpha-

alpha-




alpha-
galacturonidase A

galacturonidase




galacturonidase A


Aurpu2p4_008807
AURPU_3_00341
unknown
unknown
Hemicellulose-
unknown GH4317
GH43

607
608
609
1976
2363
2750






modfiying


Aurpu2p4_008838

unknown
unknown

unknown


610
611
612
1977
2364
2751


Aurpu2p4_008906
AURPU_3_00175
endo-
endo-
pectin-degrading
endo-
GH28

613
614
615
1978
2365
2752




polygalacturonase
polygalacturonase

polygalacturonase





GH28


Aurpu2p4_008972
Aurpu2p4_008972
Probable pectin
pectin lyase PL1
pectin-degrading
pectin lyase
PL1

616
617
618
1979
2366
2753




lyase A


Aurpu2p4_008980
AURPU_3_00147
Probable beta-
beta-mannosidase
hemicellulose-
beta-mannosidase
GH2

619
620
621
1980
2367
2754




mannosidase A
GH2
degrading


Aurpu2p4_009032

Carboxypeptidase
Carboxypeptidase S1
protein
protease


622
623
624
1981
2368
2755




S1 homolog B
homolog B
hydrolysis


Aurpu2p4_009051
Aurpu2p4_009051
Cellobiose
Cellobiose
lignin-degrading
cellobiose


625
626
627
1982
2369
2756




dehydrogenase
dehydrogenase

dehydrogenase


Aurpu2p4_009071
AURPU_3_00110
unknown
unknown

unknown GH16
GH16

628
629
630
1983
2370
2757


Aurpu2p4_009125

Cytosolic
Cytosolic
Phospholipid-
lipase


631
632
633
1984
2371
2758




phospholipase A2
phospholipase A2
modifying


Aurpu2p4_009223
Aurpu2p4_009223
Alpha-fucosidase A
Alpha-fucosidase A
Carbohydrate-
Alpha-fucosidase
GH95

634
635
636
1985
2372
2759






modifying


Aurpu2p4_009233
AURPU_3_00016
Endo-1,4-beta-
tomatinase GH10
Tomatin
tomatinase
GH10

637
638
639
1986
2373
2760




xylanase C

degrading


Aurpu2p4_009300
AURPU_3_00153
Beta-
Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH2

640
641
642
1987
2374
2761




glucuronidase

degrading


Aurpu2p4_009394
Aurpu2p4_009394
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase
CE1

643
644
645
1988
2375
2762




esterase B-1

modfiying


Aurpu2p4_009401
Aurpu2p4_009401
unknown
unknown

unknown CE5
CE5

646
647
648
1989
2376
2763


Aurpu2p4_009472

unknown
Unsaturated
pectin-degrading
rhamno-
GH105

649
650
651
1990
2377
2764





rhamnogalacturonyl

galacturonyl





hydrolase YteR

hydrolase


Aurpu2p4_009494

Lipase B
Lipase B
Lipid-degrading
lipase


652
653
654
1991
2378
2765


Aurpu2p4_009495
AURPU_3_00410
arabinoxylan
arabinoxylan
hemicellulose-
arabino-
GH62

655
656
657
1992
2379
2766




arabino-
arabino-furanosidase
degrading
furanosidase18




furanosidase
GH62


Aurpu2p4_009496
AURPU_3_00333
Beta-xylosidase
Xylosidase/arabinosidase
Hemicellulose-
Xylosidase/
GH43

658
659
660
1993
2380
2767






degrading
arabinosidase


Aurpu2p4_009563

Adhesin protein,
possible adhesin

adhesin


661
662
663
1994
2381
2768




putative


Aurpu2p4_009597
Aurpu2p4_009597
unknown
GDSL esterase/lipase
Lipid-degrading
lipase
CE16

664
665
666
1995
2382
2769





EXL5


Aurpu2p4_009603

Expansin family
unknown
Cellulase-
expansin


667
668
669
1996
2383
2770




protein

enhancing


Aurpu2p4_009751
Aurpu2p4_009751
xylanase
Xylanase GH10
hemicellulose-
xylanase
GH10

670
671
672
1997
2384
2771






degrading


Aurpu2p4_009762
Aurpu2p4_009762
endo-
endo-
pectin-degrading
rhamno-
GH28

673
674
675
1998
2385
2772




rhamnogalacturonase
rhamnogalacturonase

galacturonase





GH28


Aurpu2p4_009775

Glucoamylase
Glucoamylase
starch-
Glucoamylase


676
677
678
1999
2386
2773






degrading


Aurpu2p4_009782
AURPU_3_00011
Beta-glucosidase
beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1

679
680
681
2000
2387
2774




26

degrading


Aurpu2p4_009845
AURPU_3_00402
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61
CBM1
682
683
684
2001
2388
2775




enhancing
monooxygenase
degrading
monooxygenase




protein


Aurpu2p4_009863
AURPU_3_00105
unknown
unknown

unknown GH16
GH16

685
686
687
2002
2389
2776


Aurpu2p4_009889
Aurpu2p4_009889
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase


688
689
690
2003
2390
2777




esterase B-2

modifying


Aurpu2p4_009890
Aurpu2p4_009890
Probable beta-
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

691
692
693
2004
2391
2778




glucosidase D

degrading


Aurpu2p4_009910
AURPU_3_00219
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

694
695
696
2005
2392
2779






degrading


Aurpu2p4_010058
Aurpu2p4_010058
Probable alpha-
alpha-galactosidase
hemicellulose-
alpha-
GH27
CBM35
697
698
699
2006
2393
2780




galactosidase D
GH27
degrading
galactosidase


Aurpu2p4_010070
Aurpu2p4_010070
beta-glucosidase
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

700
701
702
2007
2394
2781






degrading


Aurpu2p4_010087
AURPU_3_00339
Xylosidase/arabinosidase
Xylosidase/arabinosidase
hemicellulose-
Xylosidase/
GH43

703
704
705
2008
2395
2782






modifying
arabinosidase


Aurpu2p4_010088
Aurpu2p4_010088
alpha-
alpha-glucuronidase
hemicellulose-
alpha-
GH67

706
707
708
2009
2396
2783




glucuronidase
GH67
degrading
glucuronidase


Aurpu2p4_010125
AURPU_3_00407
cellulase-
polysaccharide
cellulose-
polysaccharide
GH61

709
710
711
2010
2397
2784




enhancing
monooxygenase
degrading
monooxygenase




protein


Aurpu2p4_010146

Tripeptidyl-
Tripeptidyl-peptidase
Protein
protease


712
713
714
2011
2398
2785




peptidase sed4
sed4
hydrolysis


Aurpu2p4_010192
Aurpu2p4_010192
Probable beta-
Beta-galactosidase
hemicellulose-
beta-galactosidase
GH35

715
716
717
2012
2399
2786




galactosidase B
GH35
degrading


Aurpu2p4_010196
AURPU_3_00340
Putative beta-
arabinoxylan
hemicellulose-
arabino-
GH43

718
719
720
2013
2400
2787




xylosidase
arabinofuranohydrolase
degrading
furanosidase





GH43


Aurpu2p4_010203
Aurpu2p4_010203
Rhamnogalacturonan
rhamnogalacturonan
pectin-degrading
rhamno-
CE12

721
722
723
2014
2401
2788




acetylesterase
acetylesterase CE12

galacturonan







acetylesterase


Aurpu2p4_010291
Aurpu2p4_010291
Probable pectate
pectate lyase PL3
pectin-degrading
pectate lyase
PL3

724
725
726
2015
2402
2789




lyase E


Aurpu2p4_010300
AURPU_3_00015
beta-mannanase
beta-mannanase GH5
Hemicellulose-
beta-mannanase
GH5
CBM1
727
728
729
2016
2403
2790






degrading


Aurpu2p4_010313
Aurpu2p4_010313
Chitin deacetylase
Bifunctional
hemicellulose-
bifunctional
CE4
CBM18
730
731
732
2017
2404
2791





xylanase/deacetylase
degrading
xylanase/







deacetylase


Aurpu2p4_010319
Aurpu2p4_010319
Laccase-2
Laccase-2
lignin-degrading
laccase


733
734
735
2018
2405
2792


Aurpu2p4_010388
Aurpu2p4_010388
Putative
exo-polygalacturonase
pectin-degrading
exo-
GH28

736
737
738
2019
2406
2793




galacturan 1,4-
GH28

polygalacturonase




alpha-




galacturonidase C


Aurpu2p4_010455
AURPU_3_00312
Probable glucan
exo-1,3-beta-
glucan-
exo-1,3-beta-
GH5

739
740
741
2020
2407
2794




1,3-beta-
glucanase GH5
degrading
glucanase




glucosidase A


Aurpu2p4_010457
AURPU_3_00408
unknown
unknown
cellulose-
polysaccharide
GH61

742
743
744
2021
2408
2795






degrading
monooxygenase


Aurpu2p4_010464
Aurpu2p4_010464
Rhamnogalacturonate
Rhamnogalacturonate
pectin-degrading
rhamno-
PL4

745
746
747
2022
2409
2796




lyase
lyase

galacturonase


Aurpu2p4_010466
Aurpu2p4_010466
Acetylxylan
Acetylxylan esterase 2
hemicellulose-
acetylxylan
CE5

748
749
750
2023
2410
2797




esterase 2
CE5
degrading
esterase


Aurpu2p4_010484

Leucine
Aminopeptidase Y
Protein
protease


751
752
753
2024
2411
2798




aminopeptidase 2

hydrolysis


Aurpu2p4_010534
Aurpu2p4_010534
Probable pectate
pectate lyase PL1
pectin-degrading
pectate lyase
PL1

754
755
756
2025
2412
2799




lyase A


Aurpu2p4_010571
AURPU_3_00294
unknown
unknown

unknown GH43
GH43

757
758
759
2026
2413
2800


Aurpu2p4_010592

Alkaline proteinase
Alkaline protease 1
Protein
protease


760
761
762
2027
2414
2801






hydrolysis


Aurpu2p4_010596
Aurpu2p4_010596
Probable pectate
pectate lyase PL1
pectin-degrading
pectate lyase
PL1

763
764
765
2028
2415
2802



lyase A


Aurpu2p4_010603
Aurpu2p4_010603
Probable glucan
Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5

766
767
768
2029
2416
2803




1,3-beta-
beta-glucosidase A
degrading
glucosidase A




glucosidase A


Aurpu2p4_010618

Probable
Tripeptidyl-peptidase
Protein
protease


769
770
771
2030
2417
2804




tripeptidyl-
SED2
hydrolysis




peptidase SED3


Aurpu2p4_010680

Carbohydrate-
unknown

unknown


772
773
774
2031
2418
2805




binding




cytochrome b562




(Fragment)


Aurpu2p4_010683
Aurpu2p4_010683
Probable feruloyl
feruloyl esterase CE1
hemicellulose-
feruloyl esterase


775
776
777
2032
2419
2806




esterase B-1

modifying


Aurpu2p4_010701
AURPU_3_00115
unknown
unknown

unknown GH16
GH16

778
779
780
2033
2420
2807


Aurpu2p4_010884
Aurpu2p4_010884
unknown
unknown

uncharacterized


781
782
783
2034
2421
2808







lignocellulose-







induced protein


Aurpu2p4_010891

Carboxypeptidase
Carboxypeptidase S1
Protein
protease


784
785
786
2035
2422
2809




S1 homolog B
homolog A
hydrolysis


Aurpu2p4_010898
Aurpu2p4_010898
hexosaminidase
hexosaminidase GH20
chitin-degrading
hexosaminidase
GH20

787
788
789
2036
2423
2810







GH20


Aurpu2p4_010982
AURPU_3_00409
unknown
unknown
cellulose-
polysaccharide
GH61

790
791
792
2037
2424
2811






degrading
monooxygenase


Aurpu2p4_010999

Lysophospholipase 1
Lysophospholipase 2
Phospholipid-
lipase


793
794
795
2038
2425
2812






modifying


Aurpu2p4_011049
AURPU_3_00183
beta-mannanase
beta-mannanase GH5
hemicellulose-
beta-mannanase
GH5

796
797
798
2039
2426
2813






degrading


Aurpu2p4_011071
Aurpu2p4_011071
Rhamnogalacturonate
Rhamnogalacturonate
pectin-degrading
rhamno-
PL4

799
800
801
2040
2427
2814




lyase
lyase

galacturonase


Aurpu2p4_011080
AURPU_3_00240
Probable beta-
beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3

802
803
804
2041
2428
2815




glucosidase M

degrading


Aurpu2p4_011097

Aspergillopepsin-F
Aspartic protease
Protein
protease


805
806
807
2042
2429
2816





PEP1
hydrolysis


Aurpu2p4_011162

Tripeptidyl-
Tripeptidyl-peptidase
Peptide
protease


808
809
810
2043
2430
2817




peptidase sed2
sed2
hydrolysis



Aurpu2p4_000066

unknown

uncharacterized





2044
2431
2818







lignocellulose-







induced protein



Aurpu2p4_000166

Trans-1,2-

Dehydrogenase
GH109




2045
2432
2819





dihydrobenzene-1,2-





diol dehydrogenase



Aurpu2p4_000811

O-

oxidoreductase





2046
2433
2820





methylsterigmatocystin





oxidoreductase



Aurpu2p4_001233

Sterol-4-alpha-

Dehydrogenase





2047
2434
2821





carboxylate 3-





dehydrogenase,





decarboxylating



Aurpu2p4_002002

Retinol

Dehydrogenase





2048
2435
2822





dehydrogenase 10-B



Aurpu2p4_002244

Cytochrome P450

Cytochrome P450





2049
2436
2823





3A11



Aurpu2p4_002270

Tyrosinase
Pigment-
Tyrosinase





2050
2437
2824






producing



Aurpu2p4_002403

unknown

uncharacterized





2051
2438
2825







lignocellulose-







induced protein



Aurpu2p4_002547

Uncharacterized

oxidoreductase





2052
2439
2826





oxidoreductase





C26F1.07



Aurpu2p4_003458

NADPH--cytochrome

NADPH--





2053
2440
2827





P450 reductase

cytochrome P450







reductase



Aurpu2p4_003964

unknown

uncharacterized





2054
2441
2828







lignocellulose-







induced protein



Aurpu2p4_004483

Uncharacterized

oxidoreductase





2055
2442
2829





oxidoreductase dltE



Aurpu2p4_004802

O-

oxidoreductase





2056
2443
2830





methylsterigmatocystin





oxidoreductase



Aurpu2p4_005858

unknown

uncharacterized
GH128




2057
2444
2831







lignocellulose-







induced protein



Aurpu2p4_006413

Saccharopine

Dehydrogenase





2058
2445
2832





dehydrogenase





[NADP(+), L-





glutamate-forming]



Aurpu2p4_007081

Tannase
Tannin-
tannase





2059
2446
2833






degrading



Aurpu2p4_007695

unknown

unknown GH16
GH16




2060
2447
2834



Aurpu2p4_008408

unknown

uncharacterized





2061
2448
2835







lignocellulose-







induced protein



Aurpu2p4_008733

Carboxylesterase 4A

carboxylesterase
CE10




2062
2449
2836



Aurpu2p4_009064

unknown

uncharacterized





2063
2450
2837







lignocellulose-







induced protein



Aurpu2p4_009608

unknown

uncharacterized





2064
2451
2838







lignocellulose-







induced protein



Aurpu2p4_009911

Liver carboxylesterase 1

carboxylesterase
CE10




2065
2452
2839



Aurpu2p4_009938

unknown

uncharacterized





2066
2453
2840







lignocellulose-







induced protein



Aurpu2p4_010261

unknown

uncharacterized





2067
2454
2841







lignocellulose-







induced protein



Aurpu2p4_010853

Cytochrome P450 1A1

Cytochrome P450





2068
2455
2842



Aurpu2p4_011048

unknown

uncharacterized





2069
2456
2843







lignocellulose-







induced protein



AURPU_00050

unknown

unknown CE5
CE5




2070
2457
2844



AURPU_00052

Xylanase GH10
hemicellulose-
xylanase
GH10




2071
2458
2845






degrading



AURPU_00075

alpha-
hemicellulose-
alpha-
GH51




2072
2459
2846





arabinofuranosidase
degrading
arabinofuranosidase





GH51



AURPU_00077

Pectinesterase
pectin-degrading
Pectinesterase
CE8




2073
2460
2847



AURPU_00104

Cutinase
cutin-degrading
Cutinase
CE5




2074
2461
2848



AURPU_00109

Cellobiose
lignin-degrading
Cellobiose





2075
2462
2849





dehydrogenase

dehydrogenase



AURPU_00159

Probable glycosidase
Carbohydrate-
glycosidase
GH16




2076
2463
2850





crf1
modifying



AURPU_00163

Polysaccharide
cellulose-
polysaccharide
GH61




2077
2464
2851





monooxygenase
degrading
monooxygenase



AURPU_00174

Tyrosinase
Pigment-
Tyrosinase





2078
2465
2852






producing



AURPU_00225

Endo-
pectin-degrading
endo-
GH28




2079
2466
2853





polygalacturonase

polygalacturonase





GH28



AURPU_00252

unknown

unknown





2080
2467
2854



AURPU_00265

xylanase GH10
hemicellulose-
xylanase
GH10




2081
2468
2855






degrading



AURPU_00300

xyloglucanase GH12
hemicellulose-
xyloglucanase
GH12




2082
2469
2856






degrading



AURPU_00305

Putative
cellulose-
endoglucanase
GH45




2083
2470
2857





endoglucanase type K
degrading



AURPU_00319

arabinogalactanase
hemicellulose-
arabino-
GH53




2084
2471
2858





GH53
degrading
galactanase



AURPU_00344

Endo-beta-1,4-
Glucan-
endo-beta-1,4-
GH5




2085
2472
2859





glucanase A
degrading
glucanase



AURPU_00374

xylanase GH11
hemicellulose-
xylanase
GH11




2086
2473
2860






degrading



AURPU_00399

unknown

unknown





2087
2474
2861



AURPU_00402

exo-1,3-beta-
cellulose-
exo-1,3-beta-
GH5




2088
2475
2862





glucanase GH5
degrading
glucanase



AURPU_00430

unknown

unknown CE5
CE5




2089
2476
2863



AURPU_00431

Acetylxylan esterase 1
hemicellulose-
acetylxylan
CE1




2090
2477
2864





CE1
degrading
esterase



AURPU_00439

Polysaccharide
cellulose-
Polysaccharide
GH61
CBM1



2091
2478
2865





monooxygenase
degrading
monooxygenase





GH61



AURPU_00457

Probable beta-
cellulose-
Beta-glucosidase
GH3




2092
2479
2866





glucosidase A
degrading



AURPU_00470

xylanase GH11
hemicellulose-
xylanase
GH11




2093
2480
2867






degrading



AURPU_00475

Probable endo-1,3(4)-
cellulose-
endo-1,3(4)-beta-
GH16




2094
2481
2868





beta-glucanase
degrading
glucanase





AFUA_2G14360



AURPU_00476

Probable endo-1,3(4)-
cellulose-
endo-1,3(4)-beta-
GH16




2095
2482
2869





beta-glucanase
degrading
glucanase





NFIA_089530



AURPU_2_00209

Alpha-N-
hemicellulose-
Alpha-N-arabino-
GH43




2096
2483
2870





arabinofuranosidase 2
degrading
furanosidase



AURPU_2_00581

Probable
pectin-degrading
exopoly-
GH28




2097
2484
2871





exopolygalacturonase B

galacturonase



AURPU_2_01541

xylanase GH11
hemicellulose-
xylanase
GH11




2098
2485
2872






degrading



AURPU_2_01594

beta-glucosidase GH3
cellulose-
beta-glucosidase
GH3




2099
2486
2873






degrading



AURPU_2_02646

unknown

uncharacterized





2100
2487
2874







lignocellulose-







induced protein



AURPU_2_03623

Endo-
pectin-degrading
Endo-
GH28




2101
2488
2875





polygalacturonase

polygalacturonase





GH28



AURPU_2_04552

Xylanase GH10
hemicellulose-
Xylanase
GH10




2102
2489
2876






degrading



AURPU_2_04949

Probable glycosidase
Carbohydrate-
glycosidase
GH16




2103
2490
2877





crf1
modifying



AURPU_2_05877

Polysaccharide
cellulose-
Polysaccharide
GH61




2104
2491
2878





monooxygenase
degrading
monooxygenase



AURPU_2_06264

arabinoxylan
hemicellulose-
arabinoxylan
GH43




2105
2492
2879





arabinofuranohydrolase
degrading
arabinofurano-





GH43

hydrolase



AURPU_3_00001

beta-glucosidase GH1
cellulose-
beta-glucosidase
GH1




2106
2493
2880






degrading



AURPU_3_00014

Xylanase GH10
hemicellulose-
Xylanase
GH10




2107
2494
2881






degrading



AURPU_3_00018

xylanase GH11
hemicellulose-
xylanase19
GH11




2108
2495
2882






degrading



AURPU_3_00023

Endo-1,4-beta-
hemicellulose-
Endo-1,4-beta-
GH11




2109
2496
2883





xylanase B
degrading
xylanase



AURPU_3_00024

Endo-1,4-beta-
hemicellulose-
Endo-1,4-beta-
GH11




2110
2497
2884





xylanase B
degrading
xylanase



AURPU_3_00051

unknown

unknown GH16
GH16




2111
2498
2885



AURPU_3_00113

Probable glycosidase
Carbohydrate
glycosidase
GH16




2112
2499
2886





crf1
modifying



AURPU_3_00118

Probable glycosidase
Carbohydrate-
glycosidase
GH16
CBM18



2113
2500
2887





crf2
modifying



AURPU_3_00139

Chitinase GH18
chitin-degrading
Chitinase
GH18




2114
2501
2888



AURPU_3_00156

Endo-
pectin-degrading
Endo-rhamno-
GH28




2115
2502
2889





rhamnogalacturonase

galacturonase





GH28



AURPU_3_00173

exo-polygalacturonase
pectin-degrading
exo-
GH28




2116
2503
2890





GH28

polygalacturonase



AURPU_3_00174

exo-
pectin-degrading
exo-rhamno-
GH28




2117
2504
2891





rhamnogalacturonase

galacturonase





GH28



AURPU_3_00208

beta-glucosidase
cellulose-
beta-glucosidase
GH3




2118
2505
2892





GH3
degrading



AURPU_3_00209

Probable beta-
cellulose-
Beta-glucosidase
GH3




2119
2506
2893





glucosidase D
degrading



AURPU_3_00307

Beta-galactosidase
hemicellulose-
Beta-galactosidase
GH35




2120
2507
2894





GH35
degrading



AURPU_3_00428

Probable alpha-
hemicellulose-
Alpha-
GH67




2121
2508
2895





glucuronidase A
degrading
glucuronidase



Aurpu2p4_000157

Probable serine
Protein
protease





2122
2509
2896





protease EDA2
hydrolysis



Aurpu2p4_000356

Putative
lignin-degrading
peroxidase





2123
2510
2897





sterigmatocystin





biosynthesis





peroxidase stcC



Aurpu2p4_000818

unknown

unknown GH121
GH121




2124
2511
2898



Aurpu2p4_000960

Lipase 1
Lipid-degrading
lipase
CE10




2125
2512
2899



Aurpu2p4_001076

Lipase 4
Lipid-degrading
lipase
CE10




2126
2513
2900



Aurpu2p4_001476

feruloyl esterase CE1
hemicellulose-
feruloyl esterase





2127
2514
2901






degrading



Aurpu2p4_001745

possible hydrophobin

hydrophobin





2128
2515
2902



Aurpu2p4_001987

Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5




2129
2516
2903





beta-glucosidase A
degrading
glucosidase



Aurpu2p4_002339

Lipase 2
Lipid-degrading
lipase
CE10




2130
2517
2904



Aurpu2p4_002490

Minor extracellular
Protein
protease





2131
2518
2905





protease vpr
hydrolysis



Aurpu2p4_002528

Probable aspartic-type
Protein
protease





2132
2519
2906





endopeptidase OPSB
hydrolysis



Aurpu2p4_003052

Gluconolactonase

gluconolactonase





2133
2520
2907



Aurpu2p4_003108

unknown

unknown CE1
CE1




2134
2521
2908



Aurpu2p4_003243

possible pyranose
Sugar-modifying
pyranose





2135
2522
2909





dehydrogenase

dehydrogenase



Aurpu2p4_003247

possible pyranose
Sugar-modifying
pyranose





2136
2523
2910





dehydrogenase

dehydrogenase



Aurpu2p4_003704

Neutral protease 2
Protein
protease





2137
2524
2911





homolog
hydrolysis





SNOG_10522



Aurpu2p4_004187

Probable glucan 1,3-
cellulose-
glucan 1,3-beta-
GH5




2138
2525
2912





beta-glucosidase A
degrading
glucosidase



Aurpu2p4_004476

Probable endo-1,3(4)-
cellulose-
endo-1,3(4)-beta-
GH16




2139
2526
2913





beta-glucanase
degrading
glucanase





ACLA_073210



Aurpu2p4_004865

Extracellular
Protein
protease





2140
2527
2914





metalloprotease
hydrolysis





AO090012001025



Aurpu2p4_005304

Uncharacterized

unknown CE7
CE7




2141
2528
2915





protein PA2218



Aurpu2p4_005861

WSC domain-

unknown CE1
CE1




2142
2529
2916





containing protein 1



Aurpu2p4_005992

possible adhesin

adhesin





2143
2530
2917



Aurpu2p4_006091

Probable aspartic-type
Protein
protease





2144
2531
2918





endopeptidase
hydrolysis





AFUA_3G01220



Aurpu2p4_006277

Lipase 1
Lipid-degrading
lipase
CE10




2145
2532
2919



Aurpu2p4_007520

Gluconolactonase

gluconolactonase





2146
2533
2920



Aurpu2p4_007546

possible adhesin

adhesin





2147
2534
2921



Aurpu2p4_007951

Lipase 1
Lipid-degrading
lipase
CE10




2148
2535
2922



Aurpu2p4_008628

Lipase 4
Lipid-degrading
lipase
CE10




2149
2536
2923



Aurpu2p4_008719

Putative
lignin-degrading
peroxidase





2150
2537
2924





sterigmatocystin





biosynthesis





peroxidase stcC



Aurpu2p4_009254

Lipase 1
Lipid-degrading
lipase
CE10




2151
2538
2925



Aurpu2p4_009278

Lipase 1
Lipid-degrading
lipase
CE10




2152
2539
2926



Aurpu2p4_009437

possible pyranose
Sugar-modifying
pyranose





2153
2540
2927





dehydrogenase

dehydrogenase



Aurpu2p4_009445

Lipase 2
Lipid-degrading
lipase
CE10




2154
2541
2928



Aurpu2p4_010136

unknown

unknown GH2
GH2




2155
2542
2929



Aurpu2p4_010244

Lipase 1
Lipid-degrading
lipase
CE10




2156
2543
2930



Aurpu2p4_010617

Uncharacterized

unknown CE7
CE7




2157
2544
2931





protein PA2218



Aurpu2p4_010719

Bacilysin biosynthesis

oxidoreductase





2158
2545
2932





oxidoreductase BacC



Aurpu2p4_010798

Lipase 1

Lipid-degrading
lipase
CE10



2159
2546
2933



Aurpu2p4_010869

possible hydrophobin

hydrophobin





2160
2547
2934






10For example, endo-1,4-beta-xylanase.




11For example, cellulose 1,4-beta-cellobiosidase




12For example, alpha-N-arabinofuranosidase




13Probable arabinosidase or beta-galactanase.




14For example, xylan 1,4-beta-xylanase




15For example, endo-1,4-beta-xylanase.




16For example, endo-1,4-beta-xylanase.




17Demonstrates arabinosidase or arabino(furano)sidases activity (see Example 22).




18For example, alpha-L-arabinofuranosidase axhA-1




19For example, endo-1,4-beta-xylanase














TABLE 2A







List of genes of Scytalidium thermophilum with reference to exon boundaries











Genomic
Genomic




sequence
sequence


Gene ID
(SEQ ID NO:)
length
Exon boundaries (nucleotide positions) and exons













Scyth2p4_000006
1
1405
1 . . . 83, 138 . . . 541, 633 . . . 679, 799 . . . 1212, 1271 . . . 1405


Scyth2p4_000010
2
964
1 . . . 178, 246 . . . 964


Scyth2p4_000016
3
1809
1 . . . 161, 234 . . . 894, 968 . . . 1019, 1077 . . . 1555, 1627 . . . 1809


Scyth2p4_000019
4
656
1 . . . 310, 385 . . . 656


Scyth2p4_000123
5
1677
1 . . . 1677


Scyth2p4_000124
6
873
1 . . . 78, 179 . . . 312, 373 . . . 529, 622 . . . 873


Scyth2p4_000141
7
1560
1 . . . 1120, 1184 . . . 1560


Scyth2p4_000168
8
971
1 . . . 261, 327 . . . 971


Scyth2p4_000230
9
1325
1 . . . 1073, 1145 . . . 1325


Scyth2p4_000277
10
2072
1 . . . 204, 280 . . . 1652, 1712 . . . 2072


Scyth2p4_000610
11
1515
1 . . . 421, 503 . . . 1515


Scyth2p4_000863
12
1946
1 . . . 165, 280 . . . 1446, 1518 . . . 1946


Scyth2p4_000904
13
1332
1 . . . 1332


Scyth2p4_001035
14
2348
1 . . . 301, 417 . . . 547, 640 . . . 906, 972 . . . 2348


Scyth2p4_001183
15
1728
1 . . . 493, 563 . . . 1728


Scyth2p4_001259
16
652
1 . . . 360, 428 . . . 652


Scyth2p4_001262
17
1331
1 . . . 360, 440 . . . 1184, 1270 . . . 1331


Scyth2p4_001326
18
988
1 . . . 423, 491 . . .633, 691 . . . 988


Scyth2p4_001371
19
2659
1 . . . 191, 257 . . . 560, 623 . . . 997, 1061 . . . 2659


Scyth2p4_001379
20
1751
1 . . . 208, 287 . . . 545, 612 . . . 1356, 1418 . . . 1609, 1689 . . . 1751


Scyth2p4_001450
21
1451
1 . . . 43, 112 . . . 974, 1065 . . . 1451


Scyth2p4_001460
22
1721
1 . . . 323, 388 . . . 486, 538, 776, 847 . . . 1721


Scyth2p4_001513
23
5221
1 . . . 615, 680 . . . 782, 836 . . . 894, 987 . . . 1272, 1334 . . . 1399, 1467 . . . 1552,





1622 . . . 2274, 2344 . . . 2498, 2557 . . . 2719, 2776 . . . 2818, 2881 . . .3232,





3297 . . . 3686, 3791 . . . 4397, 4492 . . . 5221


Scyth2p4_001745
24
1251
1 . . . 1251


Scyth2p4_001867
25
2739
1 . . . 639, 691 . . .2739


Scyth2p4_001875
26
1504
1 . . . 331, 397 . . . 629, 689, 797, 854 . . . 1135, 1194 . . . 1504


Scyth2p4_001878
27
1185
1 . . . 342, 396 . . . 683, 745 . . . 1093, 1148 . . . 1185


Scyth2p4_001887
28
6439
1 . . . 135, 191 . . . 1674, 2408 . . . 2497, 2576 . . . 2647, 2710 . . . 3205, 3965 . . . 4543,





5548 . . . 5672, 5743 . . . 6439


Scyth2p4_001903
29
1287
1 . . . 210, 262 . . . 425, 483 . . . 1143, 1204 . . . 1287


Scyth2p4_001974
30
1340
1 . . . 745, 817 . . . 1340


Scyth2p4_001995
31
859
1 . . . 104, 161 . . . 731, 788 . . . 859


Scyth2p4_001998
32
867
1 . . . 867


Scyth2p4_002014
33
1285
1 . . . 271, 348 . . . 438, 497 . . . 542, 607 . . . 1195, 1248 . . . 1285


Scyth2p4_002032
34
908
1 . . . 489, 558 . . . 908


Scyth2p4_002058
35
2823
1 . . . 260, 375 . . . 531, 614 . . . 1151, 1216 . . . 1336, 1397 . . . 1614, 1674 . . . 2512,





2588 . . . 2694, 2775 . . . 2823


Scyth2p4_002089
36
1082
1 . . . 270, 329 . . . 919, 978 . . . 1082


Scyth2p4_002099
37
823
1 . . . 685, 744 . . . 823


Scyth2p4_002112
38
945
1 . . . 945


Scyth2p4_002143
39
2612
1 . . . 157, 209 . . . 469, 533 . . . 1140, 1188 . . . 2612


Scyth2p4_002153
40
3458
1 . . . 83, 177 . . . 200, 261 . . . 415, 3088 . . . 3458


Scyth2p4_002186
41
858
1 . . . 145, 209 . . . 858


Scyth2p4_002220
42
1104
1 . . . 105, 155 . . . 973, 1042 . . . 1104


Scyth2p4_002225
43
822
1 . . . 46, 126 . . . 414, 501 . . . 822


Scyth2p4_002425
44
927
1 . . . 186, 258 . . . 419, 517 . . . 927


Scyth2p4_002446
45
666
1 . . . 666


Scyth2p4_002491
46
3064
1 . . . 556, 618 . . . 3064


Scyth2p4_002582
47
1938
1 . . . 51, 112 . . . 1938


Scyth2p4_002596
48
1669
1 . . . 363, 425 . . . 1669


Scyth2p4_002639
49
1631
1 . . . 419, 472 . . . 557, 613 . . . 1631


Scyth2p4_002689
50
705
1 . . . 705


Scyth2p4_002854
51
1114
1 . . . 599, 664 . . . 1114


Scyth2p4_002859
52
1380
1 . . . 1380


Scyth2p4_003064
53
2677
1 . . . 290, 344 . . . 452, 519 . . . 608, 670 . . . 728, 809 . . . 838, 925 . . . 948, 1015 . . . 1074,





1140 . . . 1301, 1356 . . . 1533, 1611 . . . 1638, 1695 . . . 2237, 2297 . . . 2378,





2437 . . . 2677


Scyth2p4_003098
54
5182
1 . . . 2154, 2215 . . . 2458, 2532 . . . 2567, 2626 . . . 2592, 2947 . . . 3583, 3632 . . . 3703,





3849 . . . 3943, 4035 . . . 5182


Scyth2p4_003108
55
1621
1 . . . 166, 228 . . . 1621


Scyth2p4_003124
56
1020
1 . . . 1020


Scyth2p4_003222
57
875
1 . . . 426, 516 . . . 673, 788 . . . 875


Scyth2p4_003248
58
1992
1 . . . 1992


Scyth2p4_003738
59
3794
1 . . . 201, 308 . . . 1518, 2392 . . . 2692, 2905 . . . 3359, 3428 . . . 3794


Scyth2p4_003766
60
1338
1 . . . 552, 851 . . . 1249, 1312 . . . 1338


Scyth2p4_003836
61
2740
1 . . . 49, 116 . . . 300, 364 . . . 1791, 1856 . . . 2090, 2151 . . . 2740


Scyth2p4_003875
62
1057
1 . . . 243, 308 . . . 1057


Scyth2p4_003882
63
3067
1 . . . 81, 384 . . . 571, 624 . . . 1012, 1068 . . . 3067


Scyth2p4_003909
64
1035
1 . . . 1035


Scyth2p4_003923
65
3163
1 . . . 774, 833 . . . 3163


Scyth2p4_003925
66
1023
1 . . . 303, 365 . . . 861, 927 . . . 1023


Scyth2p4_003929
67
459
1 . . . 459


Scyth2p4_003943
68
2358
1 . . . 2358


Scyth2p4_004010
69
1479
1 . . . 127, 183 . . . 544, 613 . . . 1479


Scyth2p4_004018
70
798
1 . . . 798


Scyth2p4_004025
71
1059
1 . . . 1059


Scyth2p4_004026
72
1446
1 . . . 1446


Scyth2p4_004049
73
928
1 . . . 126, 192 . . . 285, 354 . . . 700, 764 . . . 928


Scyth2p4_004099
74
1536
1 . . . 178, 242 . . . 1536


Scyth2p4_004162
75
998
1 . . . 645, 705 . . . 998


Scyth2p4_004197
76
1607
1 . . . 90, 159 . . . 511, 576 . . . 648, 707 . . . 955, 1017 . . . 1607


Scyth2p4_004205
77
1404
1 . . . 664, 731 . . . 1404


Scyth2p4_004235
78
1324
1 . . . 1138, 1200 . . . 1324


Scyth2p4_004237
79
2264
1 . . . 1795, 1858 . . . 2264


Scyth2p4_004263
80
1586
1 . . . 628, 693 . . . 1117, 1260 . . . 1586


Scyth2p4_004293
81
1662
1 . . . 412, 473 . . . 1662


Scyth2p4_004317
82
1358
1 . . . 698, 756 . . . 1162, 1222 . . . 1358


Scyth2p4_004650
83
1474
1 . . . 419, 512 . . . 743, 823 . . . 1059, 1142 . . . 1474


Scyth2p4_004945
84
936
1 . . . 400, 510 . . . 721, 808 . . . 855, 925 . . . 936


Scyth2p4_004976
85
1476
1 . . . 317, 384 . . . 1476


Scyth2p4_005037
86
2101
1 . . . 740, 806 . . . 981, 1054 . . . 1279, 1356 . . . 1532, 1594 . . . 2101


Scyth2p4_005092
87
1032
1 . . . 34, 91 . . . 930, 998 . . . 1032


Scyth2p4_005093
88
849
1 . . . 216, 358 . . . 849


Scyth2p4_005094
89
749
1 . . . 204, 267, 749


Scyth2p4_005146
90
1437
1 . . . 195, 271 . . . 471, 531 . . . 1076, 1147 . . . 1437


Scyth2p4_005307
91
1156
1 . . . 724, 830 . . . 908, 1001 . . . 1019, 1106 . . . 1156


Scyth2p4_005334
92
1005
1 . . . 1005


Scyth2p4_005335
93
989
1 . . . 520, 586 . . . 842, 906 . . . 989


Scyth2p4_005384
94
874
1 . . . 441, 498 . . . 618, 690 . . . 874


Scyth2p4_005465
95
1101
1 . . . 958, 1031 . . . 1101


Scyth2p4_005588
96
1253
1 . . . 373, 436 . . . 1253


Scyth2p4_005596
97
2460
1 . . . 145, 220 . . . 627, 686 . . . 2460


Scyth2p4_005646
98
781
1 . . . 275, 340, 781


Scyth2p4_005692
99
893
1 . . . 320, 394, 761, 820 . . . 893


Scyth2p4_005696
100
791
1 . . . 417, 484 . . . 657, 723, 791


Scyth2p4_005712
101
1317
1 . . . 172, 229 . . . 924, 989 . . . 1317


Scyth2p4_005714
102
1394
1 . . . 1172, 1230 . . . 1305, 1371 . . . 1394


Scyth2p4_005722
103
1412
1 . . . 120, 179 . . . 278, 340 . . . 666, 752 . . . 1265, 1325 . . . 1412


Scyth2p4_005760
104
1031
1 . . . 92, 156 . . . 576, 648 . . . 1031


Scyth2p4_005775
105
478
1 . . . 183, 254 . . . 478


Scyth2p4_005777
106
477
1 . . . 198, 262 . . . 477


Scyth2p4_005792
107
2586
1 . . . 2586


Scyth2p4_005865
108
1032
1 . . . 301, 377 . . . 1032


Scyth2p4_005894
109
885
1 . . . 885


Scyth2p4_006005
110
1158
1 . . . 1158


Scyth2p4_006013
111
3539
1 . . . 334, 415 . . . 932, 1003 . . . 1182, 1254 . . . 1307, 2019 . . . 2842, 2919 . . . 3168





3280 . . . 3413, 3482 . . . 3539


Scyth2p4_006014
112
2165
1 . . . 512, 574, 752, 812 . . . 1088, 1207 . . . 1634, 1685 . . . 1814, 1883 . . . 2165


Scyth2p4_006016
113
1090
1 . . . 284, 348 . . . 681, 751 . . . 926, 994 . . . 1090


Scyth2p4_006040
114
1267
1 . . . 194, 252 . . . 1047, 1106 . . . 1267


Scyth2p4_006061
115
1755
1 . . . 621, 680 . . . 1484, 1541 . . . 1755


Scyth2p4_006263
116
1173
1 . . . 85, 141 . . . 303, 366 . . . 470, 528 . . . 1173


Scyth2p4_006265
117
2874
1 . . . 250, 406 . . . 822, 878 . . . 2874


Scyth2p4_006499
118
3030
1 . . . 82, 143 . . . 3030


Scyth2p4_006556
119
1203
1 . . . 88, 146 . . . 1203


Scyth2p4_006566
120
1244
1 . . . 348, 405 . . . 561, 622 . . . 653, 723 . . . 1244


Scyth2p4_006586
121
1092
1 . . . 1092


Scyth2p4_006591
122
2668
1 . . . 230, 290 . . . 1163, 1220 . . . 2668


Scyth2p4_006628
123
1560
1 . . . 333, 388 . . . 1560


Scyth2p4_006768
124
2631
1 . . . 79, 135 . . . 206, 273 . . . 346, 399 . . . 891, 955 . . . 2043, 2117 . . . 2631


Scyth2p4_006914
125
1687
1 . . . 1587, 1655 . . . 1687


Scyth2p4_006916
126
776
1 . . . 52, 109 . . . 385, 455 . . . 776


Scyth2p4_006920
127
1053
1 . . . 70, 150 . . . 511, 589 . . . 1053


Scyth2p4_006931
128
1105
1 . . . 423, 480 . . . 505, 562 . . . 620, 696 . . . 756, 826 . . . 1105


Scyth2p4_006993
129
1746
1 . . . 89, 148 . . . 641, 776 . . . 1395, 1450 . . . 1623, 1693 . . . 1746


Scyth2p4_007002
130
1437
1 . . . 1437


Scyth2p4_007064
131
1321
1 . . . 99, 165 . . . 318, 384 . . . 1321


Scyth2p4_007097
132
1027
1 . . . 537, 635 . . . 1027


Scyth2p4_007200
133
1465
1 . . . 297, 356, 745, 805 . . . 1160, 1231 . . . 1465


Scyth2p4_007231
134
818
1 . . . 55, 114 . . . 455, 532 . . . 818


Scyth2p4_007246
135
1179
1 . . . 140, 213 . . . 1179


Scyth2p4_007249
136
1197
1 . . . 1197


Scyth2p4_007259
137
1053
1 . . . 358, 434 . . . 1053


Scyth2p4_007263
138
888
1 . . . 448, 524 . . . 888


Scyth2p4_007266
139
3446
1 . . . 303, 368 . . . 801, 867 . . . 2167, 2224 . . . 3446


Scyth2p4_007287
140
1321
1 . . . 260, 335 . . . 497, 562 . . . 801, 856 . . . 1207, 1278 . . . 1321


Scyth2p4_007304
141
1655
1 . . . 91, 160 . . . 351, 413 . . . 479, 578 . . . 674, 731 . . . 815, 871 . . . 988, 1044 . . . 1089,





1160 . . . 1517, 1615 . . . 1655


Scyth2p4_007313
142
2402
1 . . . 191, 260 . . . 2402


Scyth2p4_007314
143
928
1 . . . 753, 812 . . . 928


Scyth2p4_007531
144
1713
1 . . . 209, 275 . . . 649, 771 . . . 928, 994 . . . 1172, 1249 . . . 1583, 1668 . . . 1713


Scyth2p4_007556
145
1062
1 . . . 1062


Scyth2p4_007557
146
1053
1 . . . 830, 1020 . . . 1053


Scyth2p4_007647
147
3340
1 . . . 819, 873 . . . 1866, 1950 . . . 3340


Scyth2p4_007651
148
844
1 . . . 46, 111 . . . 691, 758 . . . 844


Scyth2p4_007699
149
1322
1 . . . 373, 466 . . . 1322


Scyth2p4_007856
150
1104
1 . . . 1104


Scyth2p4_007921
151
1860
1 . . . 211, 265 . . . 440, 505 . . . 1860


Scyth2p4_008285
152
534
1 . . . 534


Scyth2p4_008294
153
1406
1 . . . 218, 287 . . . 435, 507 . . . 649, 706 . . . 874, 931 . . . 1406


Scyth2p4_008312
154
1508
1 . . . 201, 264 . . . 1508


Scyth2p4_008328
155
1003
1 . . . 269, 323 . . . 827, 893 . . . 1003


Scyth2p4_008336
156
1363
1 . . . 297, 358, 715, 770 . . . 1116, 1169 . . . 1363


Scyth2p4_008341
157
1066
1 . . . 58, 126 . . . 183, 236 . . . 623, 691 . . . 883, 960 . . . 1066


Scyth2p4_008344
158
595
1 . . . 181, 258 . . . 324, 391 . . . 595


Scyth2p4_008363
159
1210
1 . . . 932, 994 . . . 1210


Scyth2p4_008372
160
1583
1 . . . 570, 628 . . . 980, 1036 . . . 1279, 1336 . . . 1466, 1526 . . . 1583


Scyth2p4_008392
161
1458
1 . . . 269, 389 . . . 536, 601 . . . 797, 874 . . . 1089, 1163 . . . 1341, 1415 . . . 1458


Scyth2p4_008399
162
717
1 . . . 335, 411 . . . 717


Scyth2p4_008411
163
1377
1 . . . 784, 869 . . . 1377


Scyth2p4_008417
164
753
1 . . . 397, 470, 753


Scyth2p4_008418
165
854
1 . . . 52, 134 . . . 487, 565 . . . 854


Scyth2p4_008663
166
1293
1 . . . 22, 81 . . . 101, 222 . . . 455, 530 . . . 1293


Scyth2p4_008755
167
2176
1 . . . 316, 386 . . . 829, 895 . . . 1026, 1080 . . . 2176


Scyth2p4_008830
168
1910
1 . . . 441, 499 . . . 661, 727 . . . 1910


Scyth2p4_008896
169
1088
1 . . . 389, 496 . . . 571, 658 . . . 922, 1033 . . . 1088


Scyth2p4_009014
170
2281
1 . . . 4, 66 . . . 238, 302 . . . 565, 642 . . . 702, 769 . . . 867, 1013 . . . 1061, 1112 . . . 1167,





1218 . . . 1755, 1813 . . . 2281


Scyth2p4_009047
171
1284
1 . . . 856, 926 . . . 1284


Scyth2p4_009244
172
1742
1 . . . 219, 375 . . . 1742


Scyth2p4_009303
173
2191
1 . . . 250, 304 . . . 499, 568 . . . 2191


Scyth2p4_009308
174
1070
1 . . . 159, 246 . . . 591, 700 . . . 1070


Scyth2p4_009393
175
2615
1 . . . 83, 149 . . . 659, 733 . . . 854, 2501 . . . 2615


Scyth2p4_009418
176
1497
1 . . . 398, 462 . . . 580, 648 . . . 1192, 1264 . . . 1497


Scyth2p4_009426
177
2237
1 . . . 447, 521 . . . 642, 1874 . . . 1891, 2123 . . . 2237


Scyth2p4_009442
178
1157
1 . . . 145, 201 . . . 839, 898 . . . 1157


Scyth2p4_009463
179
1667
1 . . . 794, 853 . . . 1048, 1113 . . . 1667


Scyth2p4_009475
180
1842
1 . . . 853, 911 . . . 978, 1099 . . . 1842


Scyth2p4_009509
181
1362
1 . . . 177, 259 . . . 588, 637 . . . 1362


Scyth2p4_009510
182
1197
1 . . . 102, 151 . . . 1197


Scyth2p4_009516
183
792
1 . . . 179, 318 . . . 792


Scyth2p4_009525
184
1502
1 . . . 209, 269 . . . 426, 511 . . . 585, 669 . . . 1088, 1150 . . . 1286, 1341 . . . 1502


Scyth2p4_009550
185
1479
1 . . . 255, 312 . . . 497, 587 . . . 827, 884 . . . 1173, 1288 . . . 1479


Scyth2p4_009554
186
875
1 . . . 100, 162 . . . 523, 618 . . . 875


Scyth2p4_009565
187
1370
1 . . . 615, 678 . . . 1370


Scyth2p4_009569
188
1788
1 . . . 1042, 1105 . . . 1252, 1317 . . . 1788


Scyth2p4_009610
189
1020
1 . . . 155, 250 . . . 876, 939 . . . 1020


Scyth2p4_009620
190
974
1 . . . 335, 392 . . . 974


Scyth2p4_009626
191
1236
1 . . . 1156, 1217 . . . 1236


Scyth2p4_009629
192
1559
1 . . . 113, 186 . . . 240, 308 . . . 410, 483 . . . 616, 677 . . . 879, 935 . . . 1076, 1140 . . . 1225,





1286 . . . 1458, 1522 . . . 1559


Scyth2p4_009651
193
1407
1 . . . 1407


Scyth2p4_009653
194
1131
1 . . . 444, 521 . . . 964, 1036 . . . 1131


Scyth2p4_009700
195
1320
1 . . . 430, 513 . . . 837, 900 . . . 1320


Scyth2p4_009707
196
2834
1 . . . 49, 106 . . . 266, 325 . . . 806, 880 . . . 1219, 1275 . . . 1764, 1824 . . . 2089,





2147 . . . 2240, 2293 . . . 2525, 2592 . . . 2834


Scyth2p4_009711
197
4308
1 . . . 204, 269 . . . 1250, 2172 . . . 2881, 4132 . . . 4308


Scyth2p4_009720
198
1074
1 . . . 1074


Scyth2p4_009765
199
3723
1 . . . 23, 311 . . . 446, 1486 . . . 3723


Scyth2p4_009796
200
3125
1 . . . 235, 309 . . . 961, 1077 . . . 1160, 1240 . . . 2017, 2083 . . . 3125


Scyth2p4_009823
201
789
1 . . . 789


Scyth2p4_009929
202
4377
1 . . . 457, 506 . . . 3441, 3490 . . . 3769, 3818 . . . 4052, 4107 . . . 4377


Scyth2p4_010021
203
878
1 . . . 66, 132 . . . 436, 509 . . . 878


Scyth2p4_010034
204
1404
1 . . . 137, 204 . . . 1404


Scyth2p4_010146
205
898
1 . . . 108, 173 . . . 898


Scyth2p4_010149
206
1449
1 . . . 364, 413 . . . 521, 576, 791, 859 . . . 1349, 1403 . . . 1449


Scyth2p4_010269
207
1108
1 . . . 466, 528 . . . 1108


Scyth2p4_010278
208
5924
1 . . . 248, 315 . . . 401, 463 . . . 779, 838 . . . 924, 1001 . . . 4447, 4522 . . . 4602,





4736 . . . 5029, 5116 . . . 5214, 5353 . . . 5802, 5878 . . . 5924


Scyth2p4_010280
209
1887
1 . . . 398, 458 . . . 943, 1001 . . . 1060, 1118 . . . 1183, 1251 . . . 1755, 1810 . . . 1887


Scyth2p4_010281
210
1919
1 . . . 361, 422 . . . 503, 571 . . . 599, 673 . . . 807, 884 . . . 965, 1017 . . . 1159, 1234 . . . 1315,





1371 . . . 1585, 1667 . . . 1776, 1863 . . . 1919


Scyth2p4_010291
211
1518
1 . . . 433, 494 . . . 1518


Scyth2p4_010295
212
657
1 . . . 222, 295 . . . 657


Scyth2p4_010361
213
2163
1 . . . 181, 246 . . . 428, 529 . . . 2069, 2131 . . . 2163


Scyth2p4_010373
214
1526
1 . . . 288, 341 . . . 1085, 1144 . . . 1526


Scyth2p4_010387
215
3027
1 . . . 3027


Scyth2p4_010416
216
2030
1 . . . 1078, 1139 . . . 1914, 1986 . . . 2030


Scyth2p4_010423
217
1254
1 . . . 377, 450 . . . 1254


Scyth2p4_010457
218
815
1 . . . 278, 353 . . . 683, 741 . . . 815


Scyth2p4_010462
219
1779
1 . . . 298, 364 . . . 713, 1244 . . . 1314, 1387 . . . 1458, 1557 . . . 1779


Scyth2p4_010469
220
1917
1 . . . 592, 650 . . . 1917


Scyth2p4_010519
221
1418
1 . . . 1162, 1231 . . . 1418


Scyth2p4_010552
222
2203
1 . . . 208, 292 . . . 578, 676 . . . 802, 860 . . . 1321, 1389 . . . 2203


Scyth2p4_010553
223
2644
1 . . . 88, 1200 . . . 1752, 1831 . . . 2644


Scyth2p4_010743
224
886
1 . . . 55, 121 . . . 480, 600 . . . 886


Scyth2p4_010756
225
846
1 . . . 846


Scyth2p4_010779
226
6427
1 . . . 137, 192 . . . 449, 512 . . . 637, 710 . . . 733, 790 . . . 940, 1009 . . . 1204, 1257 . . . 1375,





1449 . . . 1654, 1742 . . . 2087, 2253 . . . 4325, 4700 . . . 6427


Scyth2p4_010780
227
2746
1 . . . 427, 481 . . . 1299, 1380 . . . 2746


Scyth2p4_010784
228
2733
1 . . . 2733


Scyth2p4_010822
229
1217
1 . . . 317, 411 . . . 803, 1124 . . . 1217


Scyth2p4_010823
230
2353
1 . . . 384, 437 . . . 2353


Scyth2p4_010825
231
1500
1 . . . 429, 484 . . . 657, 715 . . . 1500


Scyth2p4_010857
232
752
1 . . . 260, 329 . . . 752


Scyth2p4_010865
233
906
1 . . . 906


Scyth2p4_010870
234
1979
1 . . . 934, 1024 . . . 1979


Scyth2p4_010884
235
1514
1 . . . 399, 486 . . . 658, 874 . . . 1277, 1474 . . . 1514


Scyth2p4_010894
236
1143
1 . . . 369, 433 . . . 612, 712 . . . 1143


Scyth2p4_010898
237
1426
1 . . . 336, 403 . . . 629, 703 . . . 1426


Scyth2p4_010899
238
1682
1 . . . 116, 182 . . . 612, 687 . . . 1275, 1337 . . . 1682


Scyth2p4_001141
239
1447
1 . . . 112, 172 . . . 1085, 1196 . . . 1447


Scyth2p4_001257
240
1005
1 . . . 1005


Scyth2p4_001442
241
759
1 . . . 104, 201 . . . 604, 692 . . . 759


Scyth2p4_001768
242
2206
1 . . . 179, 253 . . . 912, 970 . . . 2206


Scyth2p4_002054
243
1751
1 . . . 217, 303 . . . 1130, 1195 . . . 1751


Scyth2p4_003709
244
336
1 . . . 336


Scyth2p4_003954
245
2366
1 . . . 116, 181 . . . 239, 297 . . . 335, 485 . . . 540, 595 . . . 699, 765 . . . 780, 834 . . . 1279,





1335 . . . 1657, 1720 . . . 1782, 2075 . . . 2366


Scyth2p4_004342
246
1363
1 . . . 230, 306 . . . 555, 617 . . . 1363


Scyth2p4_004817
247
1785
1 . . . 1785


Scyth2p4_005217
248
1210
1 . . . 291, 357 . . . 576, 645 . . . 1210


Scyth2p4_007345
249
1540
1 . . . 167, 233 . . . 395, 535 . . . 621, 688 . . . 851, 955 . . . 1540


Scyth2p4_007869
250
579
1 . . . 579


Scyth2p4_009477
251
2054
1 . . . 386, 445 . . . 929, 994 . . . 1811, 1887 . . . 2054


Scyth2p4_009552
252
940
1 . . . 88, 256 . . . 617, 701 . . . 940


Scyth2p4_009704
253
1311
1 . . . 1311


Scyth2p4_010302
254
551
1 . . . 141, 198 . . . 551


Scyth2p4_010820
255
565
1 . . . 208, 288 . . . 565


SCYTH_1_00385
256
2721
1 . . . 621, 673 . . . 2721


SCYTH_1_00739
257
1958
1 . . . 177, 292 . . . 1458, 1530 . . . 1958


SCYTH_1_03688
259
1059
1 . . . 1059


SCYTH_1_09019
260
1199
1 . . . 264, 333 . . . 1199


SCYTH_2_05417
261
1625
1 . . . 413, 466 . . . 551, 607 . . . 1625


Scyth2p4_000071
263
2145
1 . . . 899, 959 . . . 1427, 1519 . . . 2145


Scyth2p4_000786
264
1472
1 . . . 42, 141 . . . 298, 361 . . . 1295, 1360 . . . 1472


Scyth2p4_000879
265
1375
1 . . . 153, 218 . . . 1375


Scyth2p4_001265
266
990
1 . . . 88, 170 . . . 410, 483 . . . 757, 827 . . . 990


Scyth2p4_001349
267
2050
1 . . . 255, 331 . . . 466, 537 . . . 691, 750 . . . 1066, 1122 . . . 1297, 1360 . . . 1726,





1801 . . . 2050


Scyth2p4_002059
268
1768
1 . . . 400, 481 . . . 714, 780 . . . 971, 1045 . . . 1147, 1202 . . . 1587, 1650 . . . 1768


Scyth2p4_002062
269
1792
1 . . . 471, 530 . . . 1792


Scyth2p4_002618
270
2078
1 . . . 548, 605 . . . 2078


Scyth2p4_002885
271
1352
1 . . . 1242, 1299 . . . 1352


Scyth2p4_003845
272
2564
1 . . . 370, 442 . . . 559, 629 . . . 966, 1536 . . . 1596, 1718 . . . 1769, 2022 . . . 2564


Scyth2p4_003921
273
2399
1 . . . 2090, 2153 . . . 2399


Scyth2p4_003974
274
2051
1 . . . 305, 383 . . . 1135, 1199 . . . 2051


Scyth2p4_003996
275
2901
1 . . . 129, 196 . . . 552, 613 . . . 2901


Scyth2p4_004891
276
1507
1 . . . 333, 393 . . . 622, 676 . . . 1507


Scyth2p4_005785
277
1098
1 . . . 1098


Scyth2p4_006840
278
914
1 . . . 419, 500 . . . 914


Scyth2p4_007340
279
1926
1 . . . 111, 178 . . . 1926


Scyth2p4_007698
280
1624
1 . . . 470, 560 . . . 1094, 1154 . . . 1624


Scyth2p4_008300
281
990
1 . . . 261, 324 . . . 850, 912 . . . 990


Scyth2p4_009549
282
1648
1 . . . 295, 346 . . . 689, 752 . . . 1648


Scyth2p4_010449
283
3157
1 . . . 607, 730 . . . 1125, 1872 . . . 2083, 2138 . . . 2789, 2844 . . . 3157


Scyth2p4_010575
284
1398
1 . . . 606, 669 . . . 822, 881 . . . 1398


Scyth2p4_010881
285
1640
1 . . . 500, 602 . . . 1640
















TABLE 2B







List of genes of Myriococcum thermophilum with reference to exon boundaries











Genomic





sequence
Genomic



(SEQ
sequence


Gene ID
ID NO:)
length
Exon boundaries (nucleotide positions) and exons













Myrth2p4_000015
856
1707
1 . . . 1707


Myrth2p4_000358
857
745
1 . . . 394, 462 . . . 745


Myrth2p4_000359
858
2483
1 . . . 52, 189 . . . 1034, 1144 . . . 1265, 1394 . . . 1487, 1569 . . . 1618,





1683 . . . 1761, 1823 . . . 2055, 2129 . . . 2171, 2266 . . . 2483


Myrth2p4_000363
859
1856
1 . . . 781, 858 . . . 943, 1497 . . . 1856


Myrth2p4_000376
860
1528
1 . . . 96, 159 . . . 416, 639 . . . 835, 930 . . . 1142, 1232 . . . 1410,





1485 . . . 1528


Myrth2p4_000388
861
2404
1 . . . 561, 1278 . . . 1414, 1510 . . . 1722, 1806 . . . 2052, 2156 . . . 2286,





2347 . . . 2404


Myrth2p4_000417
862
1017
1 . . . 178, 302 . . . 1017


Myrth2p4_000486
863
732
1 . . . 732


Myrth2p4_000495
864
2067
1 . . . 261, 718 . . . 2067


Myrth2p4_000510
865
898
1 . . . 224, 352 . . . 898


Myrth2p4_000524
866
832
1 . . . 99, 188 . . . 832


Myrth2p4_000531
867
780
1 . . . 780


Myrth2p4_000543
868
1606
1 . . . 208, 276 . . . 1184, 1254 . . . 1606


Myrth2p4_000545
869
1589
1 . . . 159, 216 . . . 456, 527 . . . 1221, 1284 . . . 1368, 1423 . . . 1589


Myrth2p4_000589
870
1212
1 . . . 1212


Myrth2p4_000694
871
2139
1 . . . 204, 289 . . . 1559, 1616 . . . 1711, 1767 . . . 2139


Myrth2p4_000867
872
1534
1 . . . 442, 522 . . . 1534


Myrth2p4_000999
873
966
1 . . . 552, 733 . . . 966


Myrth2p4_001083
874
1722
1 . . . 502, 560 . . . 1722


Myrth2p4_001208
875
1570
1 . . . 192, 287 . . . 1570


Myrth2p4_001304
876
2903
1 . . . 43, 102 . . . 286, 339 . . . 1772, 1842 . . . 1984, 2042 . . . 2133,





2202 . . . 2434, 2547 . . . 2903


Myrth2p4_001319
877
908
1 . . . 644, 729 . . . 781, 859 . . . 908


Myrth2p4_001328
878
1329
1 . . . 417, 528 . . . 685, 803 . . . 1191, 1304 . . . 1329


Myrth2p4_001333
879
862
1 . . . 243, 323 . . . 862


Myrth2p4_001339
880
2984
1 . . . 72, 319 . . . 473, 534 . . . 922, 985 . . . 2984


Myrth2p4_001354
881
1147
1 . . . 79, 181 . . . 230, 290 . . . 690, 763 . . . 1147


Myrth2p4_001362
882
975
1 . . . 975


Myrth2p4_001366
883
1144
1 . . . 116, 208 . . . 1144


Myrth2p4_001368
884
2334
1 . . . 2334


Myrth2p4_001374
885
832
1 . . . 63, 156 . . . 545, 623 . . . 832


Myrth2p4_001375
886
1418
1 . . . 80, 148 . . . 548, 696 . . . 1156, 1269 . . . 1418


Myrth2p4_001378
887
1336
1 . . . 362, 445 . . . 490, 626 . . . 1211, 1305 . . . 1336


Myrth2p4_001403
888
1045
1 . . . 489, 593 . . . 1045


Myrth2p4_001451
889
1399
1 . . . 275, 982 . . . 1399


Myrth2p4_001463
890
1501
1 . . . 152, 263 . . . 411, 522 . . . 1501


Myrth2p4_001467
891
1758
1 . . . 90, 189 . . . 541, 631 . . . 703, 774 . . . 1022, 1168 . . . 1758


Myrth2p4_001469
892
1980
1 . . . 1980


Myrth2p4_001494
893
2445
1 . . . 2445


Myrth2p4_001496
894
2357
1 . . . 212, 276 . . . 1086, 1137 . . . 2357


Myrth2p4_001537
895
856
1 . . . 121, 177 . . . 301, 362 . . . 619, 674 . . . 856


Myrth2p4_001550
896
1373
1 . . . 515, 619 . . . 892, 969 . . . 1042, 1109 . . . 1281, 1336 . . . 1373


Myrth2p4_001581
897
992
1 . . . 284, 358 . . . 431, 503 . . . 544, 639 . . . 856, 936 . . . 992


Myrth2p4_001582
898
2779
1 . . . 524, 653 . . . 1227, 1324 . . . 1411, 1573 . . . 1810, 1913 . . . 1924,





2091 . . . 2221, 2500 . . . 2779


Myrth2p4_001589
899
1968
1 . . . 111, 219 . . . 1339, 1443 . . . 1870, 1943 . . . 1968


Myrth2p4_001667
900
813
1 . . . 813


Myrth2p4_001718
901
1199
1 . . . 549, 642 . . . 1199


Myrth2p4_001719
902
1137
1 . . . 602, 687 . . . 1137


Myrth2p4_001916
903
2201
1 . . . 175, 262 . . . 1542, 2161 . . . 2201


Myrth2p4_001926
904
1185
1 . . . 1185


Myrth2p4_001996
905
1648
1 . . . 432, 1188 . . . 1218, 1290 . . . 1416, 1561 . . . 1648


Myrth2p4_002010
906
2007
1 . . . 2007


Myrth2p4_002134
907
1607
1 . . . 337, 447 . . . 932, 1063 . . . 1607


Myrth2p4_002293
908
1709
1 . . . 363, 467 . . . 1211, 1648 . . . 1709


Myrth2p4_002328
909
1092
1 . . . 426, 561 . . . 703, 795 . . . 1092


Myrth2p4_002394
910
1739
1 . . . 190, 315 . . . 1318, 1376 . . . 1567, 1680 . . . 1739


Myrth2p4_002434
911
3417
1 . . . 667, 723 . . . 1169, 1240 . . . 1503, 1572 . . . 1663, 1744 . . . 1773,





1836 . . . 3071, 3172 . . . 3417


Myrth2p4_002456
912
1392
1 . . . 43, 109 . . . 965, 1048 . . . 1392


Myrth2p4_002548
913
6869
1 . . . 6781, 6853 . . . 6869


Myrth2p4_002549
914
1714
1 . . . 156, 277 . . . 735, 792 . . . 999, 1098 . . . 1452, 1522 . . . 1714


Myrth2p4_002563
915
4539
1 . . . 787, 911 . . . 943, 1060 . . . 1886, 2009 . . . 2275, 2372 . . . 3871,





3958 . . . 4539


Myrth2p4_002601
916
1392
1 . . . 1392


Myrth2p4_002632
917
2387
1 . . . 61, 165 . . . 586, 708 . . . 1379, 1500 . . . 2387


Myrth2p4_002634
918
1987
1 . . . 153, 262 . . . 1428, 1556 . . . 1987


Myrth2p4_002638
919
1431
1 . . . 109, 219 . . . 1145, 1355 . . . 1431


Myrth2p4_002915
920
1074
1 . . . 287, 381 . . . 550, 647 . . . 1074


Myrth2p4_002916
921
983
1 . . . 218, 280 . . . 632, 766 . . . 983


Myrth2p4_002917
922
1449
1 . . . 791, 936 . . . 1449


Myrth2p4_002930
923
2236
1 . . . 148, 208 . . . 838, 901 . . . 1192, 1259 . . . 1445, 1679 . . . 2052,





2213 . . . 2236


Myrth2p4_003005
924
420
1 . . . 420


Myrth2p4_003034
925
1297
1 . . . 380, 493 . . . 1297


Myrth2p4_003051
926
1461
1 . . . 388, 581 . . . 806, 868 . . . 1083, 1165 . . . 1337, 1421 . . . 1461


Myrth2p4_003065
927
1444
1 . . . 370, 512 . . . 876, 980 . . . 1053, 1136 . . . 1308, 1392 . . . 1444


Myrth2p4_003070
928
2553
1 . . . 2553


Myrth2p4_003103
929
1017
1 . . . 126, 211 . . . 304, 396 . . . 742, 853 . . . 1017


Myrth2p4_003203
930
1679
1 . . . 409, 502 . . . 1679


Myrth2p4_003274
931
1313
1 . . . 611, 722 . . . 803, 945 . . . 1313


Myrth2p4_003333
932
1614
1 . . . 259, 326 . . . 414, 490 . . . 1614


Myrth2p4_003368
933
836
1 . . . 281, 431 . . . 836


Myrth2p4_003495
934
1463
1 . . . 404, 533 . . . 764, 834 . . . 1067, 1131 . . . 1463


Myrth2p4_003633
935
953
1 . . . 406, 464 . . . 561, 631 . . . 744, 838 . . . 885, 942 . . . 953


Myrth2p4_003679
936
1377
1 . . . 1377


Myrth2p4_003685
937
1817
1 . . . 620, 721 . . . 792, 912 . . . 1183, 1264 . . . 1817


Myrth2p4_003686
938
1412
1 . . . 323, 404 . . . 1412


Myrth2p4_003747
939
880
1 . . . 152, 255 . . . 358, 448 . . . 558, 741 . . . 880


Myrth2p4_003793
940
1421
1 . . . 204, 279 . . . 479, 537 . . . 1082, 1152 . . . 1421


Myrth2p4_003921
941
1423
1 . . . 793, 997 . . . 1075, 1291 . . . 1423


Myrth2p4_003927
942
1386
1 . . . 93, 168 . . . 176, 249 . . . 281, 481 . . . 713, 814 . . . 874, 983 . . . 1235,





1343 . . . 1386


Myrth2p4_003941
943
1073
1 . . . 523, 630 . . . 871, 993 . . . 1073


Myrth2p4_003942
944
1023
1 . . . 1023


Myrth2p4_003966
945
630
1 . . . 377, 498 . . . 630


Myrth2p4_004088
946
1334
1 . . . 73, 206 . . . 368, 497 . . . 601, 683 . . . 1334


Myrth2p4_004089
947
3049
1 . . . 257, 445 . . . 872, 1021 . . . 2572, 2644 . . . 3049


Myrth2p4_004201
948
1715
1 . . . 585, 639 . . . 1443, 1498 . . . 1715


Myrth2p4_004260
949
1063
1 . . . 298, 375 . . . 641, 714 . . . 1063


Myrth2p4_004335
950
1101
1 . . . 212, 294 . . . 356, 445 . . . 495, 586 . . . 931, 994 . . . 1101


Myrth2p4_004336
951
1271
1 . . . 352, 451 . . . 1271


Myrth2p4_004345
952
2164
1 . . . 962, 1017 . . . 1035, 1114 . . . 1304, 1387 . . . 1458, 1543 . . . 2164


Myrth2p4_004391
953
1023
1 . . . 1023


Myrth2p4_004393
954
1516
1 . . . 198, 257 . . . 1516


Myrth2p4_004397
955
747
1 . . . 747


Myrth2p4_004415
956
4416
1 . . . 4416


Myrth2p4_004442
957
1619
1 . . . 328, 419 . . . 651, 723 . . . 831, 902 . . . 1183, 1315 . . . 1619


Myrth2p4_004455
958
1449
1 . . . 1449


Myrth2p4_004476
959
1406
1 . . . 153, 294 . . . 946, 1073 . . . 1242, 1360 . . . 1406


Myrth2p4_004487
960
890
1 . . . 568, 648 . . . 706, 804 . . . 890


Myrth2p4_004497
961
3374
1 . . . 837, 940 . . . 1927, 2026 . . . 3374


Myrth2p4_004508
962
922
1 . . . 98, 175 . . . 742, 821 . . . 922


Myrth2p4_004535
963
1827
1 . . . 1827


Myrth2p4_004704
964
1510
1 . . . 545, 618 . . . 792, 852 . . . 1125, 1233 . . . 1510


Myrth2p4_004725
965
2181
1 . . . 199, 267 . . . 442, 521 . . . 795, 878 . . . 1262, 1331 . . . 1937,





2129 . . . 2181


Myrth2p4_004787
966
3029
1 . . . 84, 127 . . . 337, 433 . . . 518, 582 . . . 879, 934 . . . 1022, 1141 . . . 1244,





1301 . . . 1597, 1649 . . . 3029


Myrth2p4_004788
967
1107
1 . . . 1107


Myrth2p4_004953
968
540
1 . . . 540


Myrth2p4_004960
969
1494
1 . . . 230, 336 . . . 484, 578 . . . 720, 813 . . . 981, 1043 . . . 1494


Myrth2p4_004965
970
894
1 . . . 894


Myrth2p4_004966
971
1094
1 . . . 162, 276 . . . 389, 486 . . . 1094


Myrth2p4_004986
972
1542
1 . . . 263, 942 . . . 1542


Myrth2p4_004993
973
1507
1 . . . 282, 363 . . . 548, 615 . . . 787, 861 . . . 1206, 1316 . . . 1507


Myrth2p4_005017
974
1218
1 . . . 929, 1002 . . . 1218


Myrth2p4_005025
975
4739
1 . . . 123, 186 . . . 437, 516 . . . 4739


Myrth2p4_005037
976
777
1 . . . 353, 474 . . . 777


Myrth2p4_005039
977
1352
1 . . . 1121, 1220 . . . 1352


Myrth2p4_005084
978
744
1 . . . 242, 321 . . . 744


Myrth2p4_005133
979
1232
1 . . . 156, 237 . . . 428, 503 . . . 1133, 1195 . . . 1232


Myrth2p4_005148
980
1017
1 . . . 574, 686 . . . 1017


Myrth2p4_005149
981
972
1 . . . 972


Myrth2p4_005155
982
1668
1 . . . 129, 221 . . . 320, 397 . . . 723, 916 . . . 1432, 1575 . . . 1668


Myrth2p4_005177
983
1506
1 . . . 195, 271 . . . 1506


Myrth2p4_005191
984
1232
1 . . . 468, 556 . . . 961, 1096 . . . 1232


Myrth2p4_005222
985
2622
1 . . . 2622


Myrth2p4_005269
986
1382
1 . . . 197, 281 . . . 443, 520 . . . 592, 664 . . . 1382


Myrth2p4_005317
987
3403
1 . . . 309, 389 . . . 2123, 2253 . . . 3403


Myrth2p4_005320
988
969
1 . . . 969


Myrth2p4_005321
989
972
1 . . . 466, 599 . . . 972


Myrth2p4_005328
990
1194
1 . . . 1194


Myrth2p4_005329
991
1292
1 . . . 101, 351 . . . 925, 1040 . . . 1089, 1170 . . . 1204, 1280 . . . 1292


Myrth2p4_005340
992
1649
1 . . . 393, 458 . . . 543, 622 . . . 877, 1005 . . . 1649


Myrth2p4_005343
993
975
1 . . . 55, 139 . . . 480, 611 . . . 748, 827 . . . 975


Myrth2p4_005368
994
1724
1 . . . 294, 497 . . . 886, 1077 . . . 1429, 1493 . . . 1724


Myrth2p4_005452
995
2892
1 . . . 313, 450 . . . 636, 723 . . . 2568, 2638 . . . 2892


Myrth2p4_005454
996
845
1 . . . 440, 511 . . . 607, 675 . . . 845


Myrth2p4_005463
997
1988
1 . . . 129, 199 . . . 302, 401 . . . 1067, 1856 . . . 1905, 1979 . . . 1988


Myrth2p4_005484
998
1323
1 . . . 99, 159 . . . 315, 386 . . . 1323


Myrth2p4_005539
999
2075
1 . . . 1359, 1487 . . . 1545, 1724 . . . 2075


Myrth2p4_005561
1000
1779
1 . . . 392, 450 . . . 1779


Myrth2p4_005590
1001
1462
1 . . . 1144, 1272 . . . 1462


Myrth2p4_005626
1002
1492
1 . . . 594, 730 . . . 883, 978 . . . 1492


Myrth2p4_005639
1003
1270
1 . . . 56, 123 . . . 434, 490 . . . 1270


Myrth2p4_005750
1004
892
1 . . . 55, 156 . . . 512, 606 . . . 892


Myrth2p4_005752
1005
1246
1 . . . 272, 427 . . . 1033, 1172 . . . 1246


Myrth2p4_005753
1006
1022
1 . . . 690, 831 . . . 1022


Myrth2p4_005819
1007
3131
1 . . . 303, 362 . . . 448, 527 . . . 835, 932 . . . 1920, 3032 . . . 3131


Myrth2p4_005822
1008
1563
1 . . . 179, 240 . . . 358, 470 . . . 510, 575 . . . 661, 731 . . . 921, 1029 . . . 1563


Myrth2p4_005854
1009
778
1 . . . 254, 361 . . . 778


Myrth2p4_005856
1010
1010
1 . . . 134, 258 . . . 854, 929 . . . 1010


Myrth2p4_005886
1011
1379
1 . . . 402, 512 . . . 675, 842 . . . 1236, 1330 . . . 1379


Myrth2p4_005920
1012
1712
1 . . . 113, 179 . . . 609, 679 . . . 1270, 1367 . . . 1712


Myrth2p4_005923
1013
1182
1 . . . 154, 221 . . . 344, 409 . . . 514, 590 . . . 769, 832 . . . 1182


Myrth2p4_005937
1014
1623
1 . . . 1413, 1486 . . . 1623


Myrth2p4_005945
1015
1166
1 . . . 267, 363 . . . 953, 1062 . . . 1166


Myrth2p4_005946
1016
1293
1 . . . 284, 378 . . . 445, 532 . . . 955, 1107 . . . 1293


Myrth2p4_005976
1017
1474
1 . . . 195, 261 . . . 307, 377 . . . 443, 499 . . . 556, 643 . . . 884, 982 . . . 1322,





1414 . . . 1474


Myrth2p4_006001
1018
2433
1 . . . 249, 330 . . . 408, 509 . . . 520, 630 . . . 699, 768 . . . 1749,





1857 . . . 1928, 2020 . . . 2433


Myrth2p4_006022
1019
1265
1 . . . 717, 855 . . . 1265


Myrth2p4_006028
1020
1443
1 . . . 558, 781 . . . 1443


Myrth2p4_006058
1021
967
1 . . . 320, 425 . . . 792, 894 . . . 967


Myrth2p4_006119
1022
1333
1 . . . 252, 319 . . . 954, 1088 . . . 1333


Myrth2p4_006140
1023
1107
1 . . . 1107


Myrth2p4_006141
1024
1434
1 . . . 1434


Myrth2p4_006201
1025
814
1 . . . 198, 323 . . . 814


Myrth2p4_006226
1026
1432
1 . . . 149, 231 . . . 400, 490 . . . 728, 874 . . . 1016, 1153 . . . 1432


Myrth2p4_006305
1027
1746
1 . . . 361, 438 . . . 546, 656 . . . 925, 1002 . . . 1621, 1703 . . . 1746


Myrth2p4_006387
1028
1255
1 . . . 88, 153 . . . 1255


Myrth2p4_006397
1029
2458
1 . . . 293, 356 . . . 461, 545 . . . 687, 749 . . . 833, 897 . . . 957, 1017 . . . 1130,





1221 . . . 1232, 1382 . . . 1484, 1589 . . . 2250, 2355 . . . 2458


Myrth2p4_006400
1030
1242
1 . . . 360, 425 . . . 581, 638 . . . 669, 727 . . . 1242


Myrth2p4_006403
1031
1023
1 . . . 1023


Myrth2p4_006408
1032
2597
1 . . . 248, 359 . . . 1232, 1317 . . . 1686, 1819 . . . 2597


Myrth2p4_006434
1033
1642
1 . . . 327, 440 . . . 1642


Myrth2p4_006514
1034
1662
1 . . . 372, 433 . . . 1662


Myrth2p4_006524
1035
2020
1 . . . 51, 144 . . . 1175, 1253 . . . 2020


Myrth2p4_006587
1036
3143
1 . . . 562, 658 . . . 3143


Myrth2p4_006646
1037
890
1 . . . 186, 272 . . . 433, 486 . . . 890


Myrth2p4_006765
1038
1042
1 . . . 460, 672 . . . 1042


Myrth2p4_006772
1039
836
1 . . . 49, 133 . . . 406, 515 . . . 836


Myrth2p4_006795
1040
1293
1 . . . 99, 158 . . . 304, 422 . . . 1111, 1231 . . . 1293


Myrth2p4_006807
1041
889
1 . . . 145, 243 . . . 889


Myrth2p4_006821
1042
936
1 . . . 324, 508 . . . 936


Myrth2p4_006837
1043
2207
1 . . . 192, 256 . . . 311, 380 . . . 550, 627 . . . 797, 901 . . . 1029, 1097 . . . 2207


Myrth2p4_007013
1044
648
1 . . . 209, 273 . . . 648


Myrth2p4_007061
1045
1350
1 . . . 847, 947 . . . 1350


Myrth2p4_007109
1046
2288
1 . . . 262, 315 . . . 2124, 2234 . . . 2288


Myrth2p4_007127
1047
1074
1 . . . 1074


Myrth2p4_007150
1048
947
1 . . . 122, 236 . . . 291 . . . 523 . . . 947


Myrth2p4_007367
1049
1265
1 . . . 159, 260 . . . 605, 895 . . . 1265


Myrth2p4_007409
1050
775
1 . . . 295, 408 . . . 775


Myrth2p4_007425
1051
1247
1 . . . 145, 214 . . . 852, 1000 . . . 1247


Myrth2p4_007444
1052
2844
1 . . . 49, 120 . . . 286, 343 . . . 824, 890 . . . 1229, 1309 . . . 1798,





1868 . . . 2133, 2198 . . . 2291, 2369 . . . 2844


Myrth2p4_007447
1053
1001
1 . . . 281, 398 . . . 1001


Myrth2p4_007461
1054
1398
1 . . . 439, 539 . . . 863, 978 . . . 1398


Myrth2p4_007538
1055
1748
1 . . . 1587, 1716 . . . 1748


Myrth2p4_007539
1056
1659
1 . . . 383, 509 . . . 660, 796 . . . 1218, 1395 . . . 1459, 1576 . . . 1659


Myrth2p4_007540
1057
412
1 . . . 49, 138 . . . 412


Myrth2p4_007556
1058
1334
1 . . . 414, 553 . . . 578, 671 . . . 729, 856 . . . 916, 1049 . . . 1334


Myrth2p4_007648
1059
2854
1 . . . 79, 134 . . . 205, 268 . . . 341, 408 . . . 897, 1065 . . . 2052,





2121 . . . 2188, 2337 . . . 2854


Myrth2p4_007688
1060
1185
1 . . . 993, 1090 . . . 1185


Myrth2p4_007726
1061
912
1 . . . 912


Myrth2p4_007729
1062
1343
1 . . . 168, 225 . . . 554, 612 . . . 1343


Myrth2p4_007771
1063
1668
1 . . . 108, 223 . . . 316, 461 . . . 704, 793 . . . 999, 1104 . . . 1668


Myrth2p4_007781
1064
1734
1 . . . 806, 882 . . . 1077, 1213 . . . 1734


Myrth2p4_007801
1065
1071
1 . . . 1071


Myrth2p4_007815
1066
1200
1 . . . 444, 564 . . . 1004, 1105 . . . 1200


Myrth2p4_007838
1067
1167
1 . . . 181, 292 . . . 371, 443 . . . 492, 572 . . . 616, 771 . . . 1167


Myrth2p4_007849
1068
1718
1 . . . 110, 184 . . . 238, 312 . . . 548, 652 . . . 854, 996 . . . 1137,





1198 . . . 1283, 1403 . . . 1575, 1681 . . . 1718


Myrth2p4_007850
1069
1609
1 . . . 338, 455 . . . 551, 646 . . . 845, 938 . . . 1085, 1168 . . . 1426,





1572 . . . 1609


Myrth2p4_007861
1070
1877
1 . . . 818, 928 . . . 1124, 1193 . . . 1340, 1415 . . . 1877


Myrth2p4_007867
1071
1410
1 . . . 606, 703 . . . 1410


Myrth2p4_007877
1072
884
1 . . . 100, 167 . . . 528, 639 . . . 884


Myrth2p4_007915
1073
1106
1 . . . 155, 313 . . . 930, 1022 . . . 1106


Myrth2p4_007920
1074
2385
1 . . . 255, 313 . . . 498, 1414 . . . 1496, 1564 . . . 2038, 2194 . . . 2385


Myrth2p4_007924
1075
2637
1 . . . 2637


Myrth2p4_007956
1076
753
1 . . . 753


Myrth2p4_007996
1077
2049
1 . . . 622, 701 . . . 2049


Myrth2p4_008028
1078
833
1 . . . 371, 464 . . . 833


Myrth2p4_008123
1079
1260
1 . . . 1260


Myrth2p4_008179
1080
1099
1 . . . 961, 1029 . . . 1099


Myrth2p4_008220
1081
1215
1 . . . 373, 467 . . . 1215


Myrth2p4_008285
1082
2530
1 . . . 148, 224 . . . 661, 729 . . . 2530


Myrth2p4_008298
1083
832
1 . . . 347, 445 . . . 665, 753 . . . 832


Myrth2p4_008299
1084
2568
1 . . . 203, 330 . . . 2568


Myrth2p4_008353
1085
2398
1 . . . 397, 462 . . . 2398


Myrth2p4_008360
1086
2788
1 . . . 476, 603 . . . 835, 942 . . . 2788


Myrth2p4_008429
1087
1077
1 . . . 134, 369 . . . 1077


Myrth2p4_008437
1088
903
1 . . . 903


Myrth2p4_008501
1089
1776
1 . . . 49, 165 . . . 566, 740 . . . 996, 1106 . . . 1178, 1238 . . . 1776


Myrth2p4_008515
1090
1780
1 . . . 92, 167 . . . 642, 738 . . . 1322, 1518 . . . 1780


Myrth2p4_008522
1091
1443
1 . . . 1443


Myrth2p4_008530
1092
915
1 . . . 101, 210 . . . 757, 827 . . . 915


Myrth2p4_008541
1093
918
1 . . . 918


Myrth2p4_008564
1094
727
1 . . . 307, 462 . . . 727


Myrth2p4_008615
1095
2721
1 . . . 2721


Myrth2p4_008650
1096
246
1 . . . 246


Myrth2p4_008756
1097
1296
1 . . . 1296


Myrth2p4_000413
1098
1746
1 . . . 585, 663 . . . 1034, 1108 . . . 1596, 1663 . . . 1746


Myrth2p4_000624
1099
700
1 . . . 93, 267 . . . 415, 538 . . . 700


Myrth2p4_001189
1100
2148
1 . . . 106, 160 . . . 550, 609 . . . 2148


Myrth2p4_001457
1101
1746
1 . . . 1477, 1532 . . . 1746


Myrth2p4_001536
1102
2030
1 . . . 72, 186 . . . 739, 886 . . . 1156, 1232 . . . 1514, 1580 . . . 1814,





1877 . . . 2030


Myrth2p4_001740
1103
1337
1 . . . 1218, 1290 . . . 1337


Myrth2p4_003589
1104
1593
1 . . . 378, 447 . . . 676, 735 . . . 1593


Myrth2p4_003938
1105
1474
1 . . . 257, 466 . . . 1474


Myrth2p4_006092
1106
828
1 . . . 301, 362 . . . 828


Myrth2p4_006213
1107
1376
1 . . . 651, 746 . . . 1055, 1213 . . . 1376


Myrth2p4_008350
1108
1793
1 . . . 348, 452 . . . 889, 972 . . . 1793


MYRTH_1_00009
1111
1649
1 . . . 393, 458 . . . 877, 1005 . . . 1649


MYRTH_1_00020
1112
1614
1 . . . 259, 314 . . . 418, 485 . . . 1614


MYRTH_1_00021
1113
2445
1 . . . 1323, 2275 . . . 2445


MYRTH_1_00032
1116
2544
1 . . . 801, 904 . . . 1891, 1990 . . . 2443, 2511 . . . 2544


MYRTH_1_00037
1117
815
1 . . . 660, 705 . . . 815


MYRTH_1_00069
1118
1829
1 . . . 36, 104 . . . 1270, 1398 . . . 1829


MYRTH_1_00080
1119
884
1 . . . 100, 167 . . . 528, 603 . . . 884


MYRTH_1_00084
1120
1817
1 . . . 620, 721 . . . 792, 912 . . . 1183, 1306 . . . 1817


MYRTH_1_00087
1121
3029
1 . . . 84, 127 . . . 337, 433 . . . 518, 582 . . . 1022, 1141 . . . 1244,





1301 . . . 1597, 1649 . . . 3029


MYRTH_1_00098
1122
1255
1 . . . 88, 153 . . . 460, 581 . . . 1255


MYRTH_2_00218
1123
929
1 . . . 444, 542 . . . 762, 850 . . . 929


MYRTH_2_00583
1124
3143
1 . . . 562, 658 . . . 3143


MYRTH_2_00740
1125
1290
1 . . . 1290


MYRTH_2_01076
1127
1321
1 . . . 107, 174 . . . 485, 541 . . . 1321


MYRTH_2_01077
1128
1306
1 . . . 92, 159 . . . 470, 526 . . . 1306


MYRTH_2_02633
1132
2398
1 . . . 397, 462 . . . 2398


MYRTH_2_04186
1134
1410
1 . . . 606, 703 . . . 1410


MYRTH_2_04244
1135
1218
1 . . . 929, 1002 . . . 1218


MYRTH_3_00003
1138
1343
1 . . . 168, 342 . . . 554, 612 . . . 1343


MYRTH_3_00016
1139
1839
1 . . . 1269, 1819 . . . 1839


MYRTH_3_00086
1140
1614
1 . . . 259, 314 . . . 414, 490 . . . 1614


MYRTH_3_00105
1141
1101
1 . . . 239, 294 . . . 356, 586 . . . 931, 994 . . . 1101


MYRTH_3_00120
1142
1293
1 . . . 81, 155 . . . 304, 422 . . . 1111, 1231 . . . 1293


MYRTH_3_00124
1143
1208
1 . . . 101, 351 . . . 925, 1040 . . . 1089, 1170 . . . 1208


MYRTH_4_05758
1145
1107
1 . . . 1107


MYRTH_4_09820
1147
929
1 . . . 444, 542 . . . 762, 850 . . . 929


Myrth2p4_000387
1148
2075
1 . . . 500, 590 . . . 2075


Myrth2p4_000489
1149
1882
1 . . . 505, 585 . . . 740, 816 . . . 1882


Myrth2p4_001363
1150
2403
1 . . . 2403


Myrth2p4_001546
1151
2436
1 . . . 208, 333 . . . 365, 429 . . . 694, 777 . . . 903, 970 . . . 1434,





1537 . . . 2312, 2407 . . . 2436


Myrth2p4_002267
1152
2258
1 . . . 166, 248 . . . 1162, 1268 . . . 2058, 2214 . . . 2258


Myrth2p4_002365
1153
2437
1 . . . 243, 373 . . . 508, 726 . . . 880, 1037 . . . 1353, 1454 . . . 1629,





1708 . . . 2062, 2146 . . . 2437


Myrth2p4_003086
1154
2154
1 . . . 181, 241 . . . 557, 622 . . . 2022, 2110 . . . 2154


Myrth2p4_004152
1155
840
1 . . . 840


Myrth2p4_004330
1156
1516
1 . . . 204, 280 . . . 709, 840 . . . 1516


Myrth2p4_004961
1157
1122
1 . . . 273, 371 . . . 900, 1044 . . . 1122


Myrth2p4_005807
1158
1132
1 . . . 224, 356 . . . 565, 674 . . . 831, 903 . . . 1132


Myrth2p4_005966
1159
1806
1 . . . 468, 544 . . . 1806


Myrth2p4_006645
1160
1260
1 . . . 1260


Myrth2p4_008594
1161
1622
1 . . . 1047, 1143 . . . 1622
















TABLE 2C







List of genes of Aureobasidium pullulans with reference to exon boundaries











Genomic





sequence
Genomic



(SEQ
sequence


Gene ID
ID NO:)
length
Exon boundaries (nucleotide positions) and exons













Aurpu2p4_000013
1774
1865
1 . . . 920, 1067 . . . 1865


Aurpu2p4_000017
1775
1820
1 . . . 454, 518 . . . 596, 651 . . . 858, 911 . . . 1045, 1099 . . . 1312,





1376 . . . 1681, 1735 . . . 1820


Aurpu2p4_000070
1776
1376
1 . . . 78, 132 . . . 1376


Aurpu2p4_000074
1777
2572
1 . . . 88, 153 . . . 468, 523 . . . 808, 881 . . . 2572


Aurpu2p4_000163
1778
798
1 . . . 215, 276 . . . 798


Aurpu2p4_000184
1779
1321
1 . . . 185, 247 . . . 1139, 1191 . . . 1321


Aurpu2p4_000224
1780
1215
1 . . . 288, 471 . . . 632, 687 . . . 1126, 1185 . . . 1215


Aurpu2p4_000225
1781
781
1 . . . 312, 368 . . . 781


Aurpu2p4_000232
1782
1110
1 . . . 1110


Aurpu2p4_000354
1783
1638
1 . . . 1638


Aurpu2p4_000408
1784
2361
1 . . . 2361


Aurpu2p4_000459
1785
1362
1 . . . 62, 120 . . . 200, 252 . . . 1362


Aurpu2p4_000533
1786
2403
1 . . . 2403


Aurpu2p4_000568
1787
1215
1 . . . 1215


Aurpu2p4_000586
1788
882
1 . . . 882


Aurpu2p4_000590
1789
2013
1 . . . 235, 304 . . . 648, 713 . . . 1024, 1076 . . . 2013


Aurpu2p4_000594
1790
2027
1 . . . 1250, 1298 . . . 2027


Aurpu2p4_000617
1791
1575
1 . . . 1575


Aurpu2p4_000662
1792
1380
1 . . . 279, 329 . . . 1105, 1159 . . . 1380


Aurpu2p4_000692
1793
1589
1 . . . 53, 106 . . . 767, 818 . . . 1406, 1454 . . . 1589


Aurpu2p4_000730
1794
1727
1 . . . 463, 520 . . . 1433, 1488 . . . 1727


Aurpu2p4_000792
1795
1526
1 . . . 299, 351 . . . 534, 583 . . . 646, 779 . . . 810, 954 . . . 1526


Aurpu2p4_000799
1796
2676
1 . . . 2676


Aurpu2p4_000819
1797
1791
1 . . . 329, 389 . . . 548, 625 . . . 1791


Aurpu2p4_000860
1798
2241
1 . . . 304, 353 . . . 2241


Aurpu2p4_000919
1799
1487
1 . . . 609, 661 . . . 926, 976 . . . 1091, 1142 . . . 1174, 1225 . . . 1487


Aurpu2p4_000934
1800
1381
1 . . . 446, 499 . . . 689, 741 . . . 1381


Aurpu2p4_000947
1801
1896
1 . . . 1896


Aurpu2p4_000948
1802
1863
1 . . . 171, 227 . . . 284, 339 . . . 386, 445 . . . 722, 778 . . . 1863


Aurpu2p4_000984
1803
2609
1 . . . 966, 1017 . . . 2609


Aurpu2p4_000995
1804
1104
1 . . . 455, 516 . . . 1104


Aurpu2p4_001037
1805
1828
1 . . . 43, 103 . . . 1070, 1124 . . . 1828


Aurpu2p4_001097
1806
3426
1 . . . 3426


Aurpu2p4_001104
1807
1469
1 . . . 428, 479 . . . 1469


Aurpu2p4_001152
1808
1435
1 . . . 1155, 1214 . . . 1435


Aurpu2p4_001194
1809
6009
1 . . . 6009


Aurpu2p4_001195
1810
1372
1 . . . 189, 239 . . . 651, 761 . . . 1178, 1229 . . . 1372


Aurpu2p4_001256
1811
802
1 . . . 284, 338 . . . 416, 476 . . . 802


Aurpu2p4_001441
1812
1949
1 . . . 361, 418 . . . 1949


Aurpu2p4_001503
1813
1493
1 . . . 133, 185 . . . 503, 557 . . . 1493


Aurpu2p4_001504
1814
1658
1 . . . 292, 350 . . . 517, 571 . . . 719, 773 . . . 868, 942 . . . 1163, 1224 . . . 1658


Aurpu2p4_001512
1815
1663
1 . . . 1581, 1634 . . . 1663


Aurpu2p4_001553
1816
2249
1 . . . 942, 992 . . . 1840, 1896 . . . 2144, 2199 . . . 2249


Aurpu2p4_001599
1817
1752
1 . . . 1752


Aurpu2p4_001600
1818
1728
1 . . . 1728


Aurpu2p4_001633
1819
1400
1 . . . 717, 772 . . . 1049, 1304 . . . 1400


Aurpu2p4_001665
1820
1821
1 . . . 437, 495 . . . 1821


Aurpu2p4_001680
1821
1270
1 . . . 611, 670 . . . 1270


Aurpu2p4_001713
1822
1140
1 . . . 172, 226 . . . 461, 515 . . . 713, 771 . . . 926, 980 . . . 1140


Aurpu2p4_001718
1823
1215
1 . . . 577, 639 . . . 1160, 1211 . . . 1215


Aurpu2p4_001807
1824
1125
1 . . . 1125


Aurpu2p4_001825
1825
1284
1 . . . 242, 299 . . . 416, 472 . . . 1284


Aurpu2p4_001892
1826
1245
1 . . . 1245


Aurpu2p4_001986
1827
2189
1 . . . 589, 646 . . . 2189


Aurpu2p4_002000
1828
846
1 . . . 846


Aurpu2p4_002005
1829
1173
1 . . . 1173


Aurpu2p4_002047
1830
734
1 . . . 185, 245 . . . 734


Aurpu2p4_002086
1831
1759
1 . . . 404, 454 . . . 1759


Aurpu2p4_002155
1832
850
1 . . . 316, 381 . . . 850


Aurpu2p4_002166
1833
1062
1 . . . 1062


Aurpu2p4_002167
1834
2884
1 . . . 269, 322 . . . 1074, 1129 . . . 1237, 1292 . . . 2884


Aurpu2p4_002190
1835
1310
1 . . . 102, 159 . . . 312, 364 . . . 1310


Aurpu2p4_002220
1836
1478
1 . . . 263, 312 . . . 437, 489 . . . 617, 665 . . . 731, 782 . . . 1178, 1231 . . . 1478


Aurpu2p4_002256
1837
2544
1 . . . 178, 234 . . . 932, 985 . . . 1112, 1164 . . . 1747, 1797 . . . 2544


Aurpu2p4_002267
1838
1185
1 . . . 1185


Aurpu2p4_002284
1839
1497
1 . . . 1497


Aurpu2p4_002399
1840
2713
1 . . . 146, 195 . . . 376, 427 . . . 640, 692 . . . 988, 1042 . . . 2713


Aurpu2p4_002518
1841
1485
1 . . . 427, 515 . . . 1485


Aurpu2p4_002522
1842
1079
1 . . . 351, 405 . . . 1079


Aurpu2p4_002533
1843
2256
1 . . . 177, 226 . . . 451, 501 . . . 635, 882 . . . 1744, 1811 . . . 2038,





2098 . . . 2256


Aurpu2p4_002671
1844
1110
1 . . . 1110


Aurpu2p4_002672
1845
2849
1 . . . 118, 168 . . . 263, 317 . . . 373, 430 . . . 615, 701 . . . 733, 785 . . . 874,





923 . . . 1191, 1240 . . . 1732, 1783 . . . 2849


Aurpu2p4_002750
1846
1170
1 . . . 104, 157 . . . 289, 345 . . . 484, 537 . . . 1170


Aurpu2p4_002860
1847
2220
1 . . . 2220


Aurpu2p4_002907
1848
1674
1 . . . 1674


Aurpu2p4_002940
1849
4560
1 . . . 335, 388 . . . 3884, 3938 . . . 3959, 4165 . . . 4342, 4393 . . . 4560


Aurpu2p4_002942
1850
1209
1 . . . 1209


Aurpu2p4_002955
1851
1686
1 . . . 1686


Aurpu2p4_002987
1852
1216
1 . . . 187, 241 . . . 340, 394 . . . 1216


Aurpu2p4_003029
1853
1020
1 . . . 1020


Aurpu2p4_003104
1854
1737
1 . . . 1737


Aurpu2p4_003184
1855
2442
1 . . . 969, 1017 . . . 1371, 1424 . . . 2442


Aurpu2p4_003313
1856
1809
1 . . . 334, 392 . . . 1809


Aurpu2p4_003364
1857
2784
1 . . . 2784


Aurpu2p4_003555
1858
1440
1 . . . 1440


Aurpu2p4_003594
1859
1575
1 . . . 1575


Aurpu2p4_003606
1860
1092
1 . . . 280, 332 . . . 1092


Aurpu2p4_003607
1861
1306
1 . . . 505, 562 . . . 896, 955 . . . 1113, 1166 . . . 1306


Aurpu2p4_003685
1862
881
1 . . . 245, 299 . . . 518, 570 . . . 881


Aurpu2p4_003727
1863
1468
1 . . . 542, 595 . . . 1468


Aurpu2p4_003747
1864
3552
1 . . . 212, 266 . . . 452, 709 . . . 907, 1047 . . . 1236, 1287 . . . 3552


Aurpu2p4_003884
1865
1113
1 . . . 157, 212 . . . 1113


Aurpu2p4_003888
1866
2763
1 . . . 2231, 2283 . . . 2763


Aurpu2p4_003893
1867
948
1 . . . 948


Aurpu2p4_003941
1868
1336
1 . . . 1074, 1128 . . . 1266, 1323 . . . 1336


Aurpu2p4_004107
1869
2366
1 . . . 462, 662 . . . 1591, 1644 . . . 2366


Aurpu2p4_004115
1870
1038
1 . . . 406, 467 . . . 1038


Aurpu2p4_004128
1871
1434
1 . . . 1434


Aurpu2p4_004186
1872
742
1 . . . 261, 320 . . . 742


Aurpu2p4_004265
1873
2724
1 . . . 289, 344 . . . 2724


Aurpu2p4_004286
1874
2861
1 . . . 812, 1268 . . . 1495, 1548 . . . 1575, 1627 . . . 2143, 2197 . . . 2861


Aurpu2p4_004297
1875
2145
1 . . . 1894, 1958 . . . 2145


Aurpu2p4_004347
1876
1413
1 . . . 1413


Aurpu2p4_004477
1877
2778
1 . . . 72, 124 . . . 295, 345 . . . 625, 681 . . . 836, 885 . . . 1884, 1940 . . . 2139,





2193 . . . 2356, 2406 . . . 2778


Aurpu2p4_004489
1878
2212
1 . . . 196, 465 . . . 512, 564 . . . 802, 859 . . . 1040, 1240 . . . 2212


Aurpu2p4_004524
1879
1011
1 . . . 1011


Aurpu2p4_004527
1880
2151
1 . . . 2151


Aurpu2p4_004550
1881
854
1 . . . 452, 509 . . . 854


Aurpu2p4_004694
1882
1082
1 . . . 134, 182 . . . 265, 317 . . . 1082


Aurpu2p4_004762
1883
1477
1 . . . 282, 410 . . . 536, 588 . . . 672, 726 . . . 1335, 1448 . . . 1477


Aurpu2p4_004776
1884
705
1 . . . 705


Aurpu2p4_004801
1885
2180
1 . . . 666, 715 . . . 896, 1229 . . . 1671, 1726 . . . 2180


Aurpu2p4_004899
1886
1884
1 . . . 1884


Aurpu2p4_004916
1887
1928
1 . . . 416, 475 . . . 587, 640 . . . 1928


Aurpu2p4_004926
1888
1217
1 . . . 365, 422 . . . 880, 935 . . . 1217


Aurpu2p4_004937
1889
2255
1 . . . 491, 542 . . . 918, 977 . . . 1678, 1731 . . . 1989, 2048 . . . 2255


Aurpu2p4_004986
1890
1282
1 . . . 158, 209 . . . 518, 569 . . . 1282


Aurpu2p4_005056
1891
1308
1 . . . 1308


Aurpu2p4_005097
1892
2901
1 . . . 423, 483 . . . 717, 770 . . . 2901


Aurpu2p4_005194
1893
1098
1 . . . 1098


Aurpu2p4_005236
1894
2160
1 . . . 399, 454 . . . 2160


Aurpu2p4_005278
1895
830
1 . . . 706, 763 . . . 830


Aurpu2p4_005399
1896
1036
1 . . . 327, 377 . . . 1036


Aurpu2p4_005401
1897
2397
1 . . . 120, 171 . . . 394, 443 . . . 827, 882 . . . 1226, 1283 . . . 1523,





1571 . . . 1734, 1786 . . . 2397


Aurpu2p4_005519
1898
1026
1 . . . 1026


Aurpu2p4_005580
1899
1854
1 . . . 1374, 1630 . . . 1854


Aurpu2p4_005825
1900
1220
1 . . . 413, 475 . . . 1028, 1090 . . . 1220


Aurpu2p4_005865
1901
1248
1 . . . 1248


Aurpu2p4_005914
1902
2530
1 . . . 145, 201 . . . 522, 580 . . . 2530


Aurpu2p4_005929
1903
1025
1 . . . 33, 91 . . . 333, 414 . . . 894, 958 . . . 1025


Aurpu2p4_006113
1904
1095
1 . . . 1095


Aurpu2p4_006128
1905
884
1 . . . 442, 496 . . . 884


Aurpu2p4_006160
1906
1722
1 . . . 1722


Aurpu2p4_006162
1907
1608
1 . . . 1608


Aurpu2p4_006176
1908
1484
1 . . . 445, 513 . . . 1227, 1285 . . . 1396, 1455 . . . 1484


Aurpu2p4_006179
1909
1392
1 . . . 1392


Aurpu2p4_006195
1910
2233
1 . . . 659, 711 . . . 1164, 1231 . . . 1489, 1544 . . . 1708, 1770 . . . 2233


Aurpu2p4_006206
1911
1725
1 . . . 1035, 1093 . . . 1725


Aurpu2p4_006207
1912
6091
1 . . . 203, 255 . . . 455, 508 . . . 4226, 4281 . . . 4787, 4857 . . . 5006,





5064 . . . 5293, 5339 . . . 6091


Aurpu2p4_006222
1913
1788
1 . . . 1788


Aurpu2p4_006237
1914
773
1 . . . 202, 268 . . . 773


Aurpu2p4_006246
1915
2528
1 . . . 172, 222 . . . 320, 373 . . . 2528


Aurpu2p4_006312
1916
964
1 . . . 317, 381 . . . 836, 898 . . . 964


Aurpu2p4_006313
1917
1052
1 . . . 444, 504 . . . 1052


Aurpu2p4_006392
1918
1655
1 . . . 263, 314 . . . 1655


Aurpu2p4_006557
1919
1451
1 . . . 364, 427 . . . 1451


Aurpu2p4_006782
1920
3618
1 . . . 93, 344 . . . 533, 586 . . . 592, 651 . . . 748, 801 . . . 1252, 1300 . . . 1888,





1947 . . . 2106, 2163 . . . 2336, 2390 . . . 2839, 2892 . . . 3618


Aurpu2p4_006900
1921
2341
1 . . . 416, 466 . . . 533, 582 . . . 756, 815 . . . 1135, 1186 . . . 1924,





1976 . . . 2341


Aurpu2p4_006933
1922
2242
1 . . . 278, 515 . . . 1235, 1295 . . . 2242


Aurpu2p4_007070
1923
1257
1 . . . 1257


Aurpu2p4_007082
1924
1287
1 . . . 65, 122 . . . 540, 616 . . . 1082, 1150 . . . 1287


Aurpu2p4_007093
1925
813
1 . . . 257, 308 . . . 455, 509 . . . 679, 733 . . . 813


Aurpu2p4_007113
1926
2028
1 . . . 2028


Aurpu2p4_007124
1927
777
1 . . . 360, 415 . . . 777


Aurpu2p4_007126
1928
1653
1 . . . 263, 321 . . . 1653


Aurpu2p4_007149
1929
1263
1 . . . 1263


Aurpu2p4_007160
1930
1971
1 . . . 1971


Aurpu2p4_007177
1931
2187
1 . . . 2187


Aurpu2p4_007190
1932
1865
1 . . . 289, 348 . . . 1657, 1710 . . . 1865


Aurpu2p4_007196
1933
1239
1 . . . 609, 676 . . . 1239


Aurpu2p4_007206
1934
1435
1 . . . 360, 406 . . . 573, 626 . . . 1435


Aurpu2p4_007220
1935
1089
1 . . . 538, 593 . . . 1089


Aurpu2p4_007270
1936
2669
1 . . . 118, 166 . . . 285, 333 . . . 541, 611 . . . 907, 959 . . . 1064, 1117 . . . 2669


Aurpu2p4_007272
1937
1961
1 . . . 208, 264 . . . 500, 559 . . . 617, 675 . . . 841, 902 . . . 1273, 1325 . . . 1961


Aurpu2p4_007292
1938
1064
1 . . . 71, 134 . . . 1064


Aurpu2p4_007342
1939
1042
1 . . . 77, 133 . . . 1042


Aurpu2p4_007356
1940
1433
1 . . . 723, 777 . . . 1433


Aurpu2p4_007383
1941
3123
1 . . . 3123


Aurpu2p4_007404
1942
1650
1 . . . 1650


Aurpu2p4_007424
1943
1236
1 . . . 1236


Aurpu2p4_007428
1944
1829
1 . . . 14, 430 . . . 927, 976 . . . 1739, 1795 . . . 1829


Aurpu2p4_007429
1945
1052
1 . . . 290, 350 . . . 1052


Aurpu2p4_007455
1946
1572
1 . . . 1572


Aurpu2p4_007488
1947
1131
1 . . . 228, 287 . . . 358, 418 . . . 1131


Aurpu2p4_007493
1948
1856
1 . . . 387, 568 . . . 1515, 1569 . . . 1856


Aurpu2p4_007511
1949
1152
1 . . . 253, 325 . . . 426, 480 . . . 909, 966 . . . 1152


Aurpu2p4_007612
1950
786
1 . . . 278, 330 . . . 531, 589 . . . 786


Aurpu2p4_007614
1951
1189
1 . . . 552, 602 . . . 1189


Aurpu2p4_007621
1952
1275
1 . . . 440, 496 . . . 880, 938 . . . 984, 1035 . . . 1275


Aurpu2p4_007662
1953
873
1 . . . 873


Aurpu2p4_007707
1954
843
1 . . . 132, 184 . . . 409, 461 . . . 843


Aurpu2p4_007805
1955
2072
1 . . . 1612, 1670 . . . 1851, 1911 . . . 2072


Aurpu2p4_007919
1956
895
1 . . . 109, 166 . . . 266, 317 . . . 373, 423 . . . 716, 770 . . . 895


Aurpu2p4_008001
1957
2647
1 . . . 298, 349 . . . 710, 763 . . . 2407, 2460 . . . 2647


Aurpu2p4_008021
1958
1541
1 . . . 1242, 1293 . . . 1541


Aurpu2p4_008140
1959
1636
1 . . . 369, 422 . . . 1636


Aurpu2p4_008212
1960
1203
1 . . . 338, 396 . . . 1203


Aurpu2p4_008231
1961
2251
1 . . . 1600, 1653 . . . 2251


Aurpu2p4_008239
1962
1672
1 . . . 256, 312 . . . 400, 456 . . . 1075, 1128 . . . 1215, 1274 . . . 1672


Aurpu2p4_008255
1963
880
1 . . . 701, 766 . . . 880


Aurpu2p4_008271
1964
1517
1 . . . 153, 213 . . . 1517


Aurpu2p4_008282
1965
1986
1 . . . 217, 356 . . . 1986


Aurpu2p4_008385
1966
2258
1 . . . 121, 188 . . . 871, 922 . . . 1399, 1448 . . . 2258


Aurpu2p4_008412
1967
3649
1 . . . 341, 396 . . . 478, 532 . . . 627, 742 . . . 933, 983 . . . 1180,





1279 . . . 1354, 1406 . . . 1519, 1780 . . . 3649


Aurpu2p4_008485
1968
1058
1 . . . 528, 579 . . . 1058


Aurpu2p4_008495
1969
2152
1 . . . 426, 1304 . . . 1796, 1853 . . . 2023, 2085 . . . 2152


Aurpu2p4_008503
1970
1875
1 . . . 1875


Aurpu2p4_008585
1971
2640
1 . . . 2640


Aurpu2p4_008692
1972
2039
1 . . . 117, 167 . . . 298, 354 . . . 769, 821 . . . 872, 930 . . . 2039


Aurpu2p4_008705
1973
3036
1 . . . 3036


Aurpu2p4_008725
1974
960
1 . . . 960


Aurpu2p4_008775
1975
3302
1 . . . 1356, 1653 . . . 1781, 2223 . . . 3302


Aurpu2p4_008807
1976
1129
1 . . . 310, 362 . . . 479, 532 . . . 1129


Aurpu2p4_008838
1977
2346
1 . . . 818, 1047 . . . 1484, 1539 . . . 2346


Aurpu2p4_008906
1978
1291
1 . . . 13, 64 . . . 791, 846 . . . 952, 1006 . . . 1291


Aurpu2p4_008972
1979
1941
1 . . . 258, 390 . . . 464, 664 . . . 792, 846 . . . 860, 915 . . . 983, 1040 . . . 1050,





1105 . . . 1232, 1430 . . . 1631, 1692 . . . 1941


Aurpu2p4_008980
1980
3232
1 . . . 608, 669 . . . 1379, 1531 . . . 1781, 2047 . . . 2477, 2534 . . . 3232


Aurpu2p4_009032
1981
2108
1 . . . 132, 188 . . . 410, 589 . . . 937, 1042 . . . 1650, 1703 . . . 2108


Aurpu2p4_009051
1982
2136
1 . . . 2136


Aurpu2p4_009071
1983
1397
1 . . . 336, 390 . . . 1397


Aurpu2p4_009125
1984
2549
1 . . . 50, 107 . . . 2549


Aurpu2p4_009223
1985
2464
1 . . . 154, 205 . . . 544, 595 . . . 2464


Aurpu2p4_009233
1986
1020
1 . . . 1020


Aurpu2p4_009300
1987
2697
1 . . . 2697


Aurpu2p4_009394
1988
1560
1 . . . 1560


Aurpu2p4_009401
1989
684
1 . . . 684


Aurpu2p4_009472
1990
1329
1 . . . 1329


Aurpu2p4_009494
1991
1543
1 . . . 392, 442 . . . 495, 547 . . . 1543


Aurpu2p4_009495
1992
987
1 . . . 987


Aurpu2p4_009496
1993
1746
1 . . . 1746


Aurpu2p4_009563
1994
1059
1 . . . 397, 463 . . . 968, 1018 . . . 1059


Aurpu2p4_009597
1995
1161
1 . . . 867, 925 . . . 1161


Aurpu2p4_009603
1996
800
1 . . . 311, 362 . . . 621, 679 . . . 800


Aurpu2p4_009751
1997
1437
1 . . . 452, 512 . . . 1367, 1423 . . . 1437


Aurpu2p4_009762
1998
1443
1 . . . 359, 410 . . . 702, 755 . . . 1443


Aurpu2p4_009775
1999
938
1 . . . 353, 410 . . . 938


Aurpu2p4_009782
2000
1989
1 . . . 417, 464 . . . 1041, 1092 . . . 1680, 1729 . . . 1854, 1909 . . . 1989


Aurpu2p4_009845
2001
1038
1 . . . 1038


Aurpu2p4_009863
2002
985
1 . . . 705, 762 . . . 787, 841 . . . 985


Aurpu2p4_009889
2003
1692
1 . . . 1190, 1245 . . . 1692


Aurpu2p4_009890
2004
2555
1 . . . 528, 578 . . . 869, 919 . . . 2213, 2262 . . . 2555


Aurpu2p4_009910
2005
2639
1 . . . 322, 376 . . . 2639


Aurpu2p4_010058
2006
1711
1 . . . 324, 377 . . . 1711


Aurpu2p4_010070
2007
2923
1 . . . 75, 125 . . . 320, 379 . . . 487, 593 . . . 689, 742 . . . 2032, 2082 . . . 2923


Aurpu2p4_010087
2008
1783
1 . . . 1570, 1629 . . . 1783


Aurpu2p4_010088
2009
2511
1 . . . 2511


Aurpu2p4_010125
2010
1508
1 . . . 102, 162 . . . 1508


Aurpu2p4_010146
2011
2596
1 . . . 1543, 1655 . . . 1871, 1927 . . . 2596


Aurpu2p4_010192
2012
3218
1 . . . 174, 230 . . . 297, 345 . . . 374, 429 . . . 1220, 1316 . . . 3218


Aurpu2p4_010196
2013
1599
1 . . . 477, 535 . . . 1599


Aurpu2p4_010203
2014
880
1 . . . 118, 179 . . . 413, 475 . . . 880


Aurpu2p4_010291
2015
813
1 . . . 528, 580 . . . 813


Aurpu2p4_010300
2016
1365
1 . . . 1365


Aurpu2p4_010313
2017
1937
1 . . . 262, 311 . . . 889, 941 . . . 1335, 1393 . . . 1836, 1890 . . . 1937


Aurpu2p4_010319
2018
2170
1 . . . 226, 279 . . . 535, 582 . . . 1719, 1771 . . . 1950, 2004 . . . 2170


Aurpu2p4_010388
2019
1489
1 . . . 276, 331 . . . 479, 535 . . . 758, 814 . . . 1007, 1071 . . . 1388,





1484 . . . 1489


Aurpu2p4_010455
2020
1878
1 . . . 77, 133 . . . 269, 556 . . . 813, 864 . . . 1189, 1246 . . . 1364,





1422 . . . 1663, 1717 . . . 1791, 1859 . . . 1878


Aurpu2p4_010457
2021
1581
1 . . . 1581


Aurpu2p4_010464
2022
1873
1 . . . 205, 260 . . . 708, 825 . . . 953, 1001 . . . 1873


Aurpu2p4_010466
2023
731
1 . . . 507, 558 . . . 731


Aurpu2p4_010484
2024
1749
1 . . . 292, 346 . . . 1146, 1265 . . . 1551, 1606 . . . 1749


Aurpu2p4_010534
2025
1023
1 . . . 550, 608 . . . 1023


Aurpu2p4_010571
2026
2226
1 . . . 2226


Aurpu2p4_010592
2027
1352
1 . . . 309, 364 . . . 711, 762 . . . 1352


Aurpu2p4_010596
2028
1058
1 . . . 537, 591 . . . 1058


Aurpu2p4_010603
2029
1298
1 . . . 969, 1074 . . . 1298


Aurpu2p4_010618
2030
1898
1 . . . 243, 298 . . . 606, 664 . . . 813, 867 . . . 1898


Aurpu2p4_010680
2031
1423
1 . . . 22, 90 . . . 326, 380 . . . 978, 1046 . . . 1423


Aurpu2p4_010683
2032
1596
1 . . . 275, 345 . . . 393, 446 . . . 665, 721 . . . 1452, 1565 . . . 1596


Aurpu2p4_010701
2033
1266
1 . . . 348, 402 . . . 736, 792 . . . 1266


Aurpu2p4_010884
2034
1185
1 . . . 1185


Aurpu2p4_010891
2035
2164
1 . . . 144, 236 . . . 345, 396 . . . 570, 624 . . . 943, 1003 . . . 2164


Aurpu2p4_010898
2036
1827
1 . . . 1356, 1414 . . . 1827


Aurpu2p4_010982
2037
1260
1 . . . 1260


Aurpu2p4_010999
2038
1767
1 . . . 546, 604 . . . 867, 925 . . . 1767


Aurpu2p4_011049
2039
1465
1 . . . 174, 235 . . . 561, 616 . . . 1081, 1147 . . . 1342, 1402 . . . 1465


Aurpu2p4_011071
2040
1848
1 . . . 208, 262 . . . 662, 714 . . . 764, 817 . . . 1848


Aurpu2p4_011080
2041
2451
1 . . . 127, 179 . . . 648, 699 . . . 756, 809 . . . 2451


Aurpu2p4_011097
2042
1182
1 . . . 1182


Aurpu2p4_011162
2043
1776
1 . . . 1776


Aurpu2p4_000066
2044
1899
1 . . . 1899


Aurpu2p4_000166
2045
1294
1 . . . 54, 106 . . . 519, 569 . . . 1294


Aurpu2p4_000811
2046
1683
1 . . . 138, 187 . . . 1683


Aurpu2p4_001233
2047
1583
1 . . . 901, 1459 . . . 1583


Aurpu2p4_002002
2048
1050
1 . . . 1050


Aurpu2p4_002244
2049
2043
1 . . . 221, 298 . . . 872, 929 . . . 2043


Aurpu2p4_002270
2050
1277
1 . . . 188, 242 . . . 947, 999 . . . 1277


Aurpu2p4_002403
2051
1857
1 . . . 1857


Aurpu2p4_002547
2052
1042
1 . . . 228, 278 . . . 1042


Aurpu2p4_003458
2053
2144
1 . . . 1791, 1848 . . . 2144


Aurpu2p4_003964
2054
588
1 . . . 588


Aurpu2p4_004483
2055
1073
1 . . . 441, 708 . . . 1073


Aurpu2p4_004802
2056
2161
1 . . . 124, 178 . . . 435, 486 . . . 2161


Aurpu2p4_005858
2057
1242
1 . . . 1242


Aurpu2p4_006413
2058
1557
1 . . . 35, 103 . . . 185, 246 . . . 451, 502 . . . 1557


Aurpu2p4_007081
2059
1865
1 . . . 352, 405 . . . 665, 718 . . . 1865


Aurpu2p4_007695
2060
1434
1 . . . 76, 122 . . . 666, 721 . . . 1434


Aurpu2p4_008408
2061
1127
1 . . . 71, 126 . . . 205, 261 . . . 1047, 1100 . . . 1127


Aurpu2p4_008733
2062
1835
1 . . . 723, 810 . . . 1835


Aurpu2p4_009064
2063
1113
1 . . . 1113


Aurpu2p4_009608
2064
1448
1 . . . 553, 605 . . . 1321, 1387 . . . 1448


Aurpu2p4_009911
2065
1758
1 . . . 301, 355 . . . 455, 504 . . . 606, 656 . . . 1758


Aurpu2p4_009938
2066
435
1 . . . 435


Aurpu2p4_010261
2067
1865
1 . . . 873, 927 . . . 1036, 1094 . . . 1865


Aurpu2p4_010853
2068
1654
1 . . . 219, 274 . . . 1503, 1556 . . . 1654


Aurpu2p4_011048
2069
846
1 . . . 846


AURPU_3_00014
2107
1434
1 . . . 449, 509 . . . 1364, 1420 . . . 1434


AURPU_3_00051
2111
1434
1 . . . 76, 203 . . . 666, 721 . . . 1434


AURPU_3_00113
2112
1206
1 . . . 1206


AURPU_3_00118
2113
1422
1 . . . 556, 611 . . . 1422


AURPU_3_00139
2114
1553
1 . . . 524, 861 . . . 1463, 1514 . . . 1553


AURPU_3_00156
2115
1820
1 . . . 454, 518 . . . 596, 651 . . . 825, 911 . . . 1045, 1099 . . . 1285,





1376 . . . 1681, 1735 . . . 1820


AURPU_3_00173
2116
1541
1 . . . 276, 331 . . . 479, 535 . . . 758, 814 . . . 1007, 1071 . . . 1541


AURPU_3_00174
2117
1398
1 . . . 1398


AURPU_3_00209
2119
5098
1 . . . 528, 578 . . . 869, 919 . . . 2213, 2262 . . . 2538, 2593 . . . 2634,





3540 . . . 4596, 4651 . . . 5098


AURPU_3_00307
2120
3218
1 . . . 174, 230 . . . 297, 345 . . . 374, 429 . . . 1117, 1294 . . . 3218


Aurpu2p4_000157
2122
1723
1 . . . 96, 173 . . . 1723


Aurpu2p4_000356
2123
1035
1 . . . 184, 236 . . . 1035


Aurpu2p4_000818
2124
2028
1 . . . 2028


Aurpu2p4_000960
2125
1847
1 . . . 138, 187 . . . 787, 839 . . . 906, 963 . . . 1847


Aurpu2p4_001076
2126
1797
1 . . . 445, 498 . . . 756, 807 . . . 1797


Aurpu2p4_001476
2127
1280
1 . . . 252, 309 . . . 1280


Aurpu2p4_001745
2128
417
1 . . . 184, 241 . . . 326, 382 . . . 417


Aurpu2p4_001987
2129
2335
1 . . . 425, 481 . . . 2335


Aurpu2p4_002339
2130
1860
1 . . . 264, 316 . . . 450, 512 . . . 891, 945 . . . 1860


Aurpu2p4_002490
2131
2812
1 . . . 141, 190 . . . 591, 641 . . . 932, 984 . . . 2812


Aurpu2p4_002528
2132
1532
1 . . . 364, 448 . . . 1532


Aurpu2p4_003052
2133
1275
1 . . . 1275


Aurpu2p4_003108
2134
2106
1 . . . 2106


Aurpu2p4_003243
2135
7430
1 . . . 227, 286 . . . 538, 597 . . . 729, 796 . . . 1037, 1092 . . . 1305,





1362 . . . 1454, 1504 . . . 2180, 2235 . . . 2263, 3199 . . . 3725,





3786 . . . 3809, 3869 . . . 4355, 4434 . . . 4548, 4645 . . . 4686,





4741 . . . 4788, 4848 . . . 5045, 5099 . . . 5146, 5207 . . . 5272,





5447 . . . 5518, 5583 . . . 5624, 5690 . . . 5749, 5864 . . . 5889,





6063 . . . 6093, 6196 . . . 6286, 6343 . . . 6390, 6463 . . . 6492,





6559 . . . 7035, 7090 . . . 7430


Aurpu2p4_003247
2136
2046
1 . . . 316, 365 . . . 2046


Aurpu2p4_003704
2137
1243
1 . . . 226, 283 . . . 822, 885 . . . 1166, 1224 . . . 1243


Aurpu2p4_004187
2138
2439
1 . . . 433, 485 . . . 1291, 1350 . . . 1656, 1713 . . . 2439


Aurpu2p4_004476
2139
4947
1 . . . 189, 239 . . . 267, 320 . . . 570, 620 . . . 3295, 3350 . . . 4947


Aurpu2p4_004865
2140
909
1 . . . 909


Aurpu2p4_005304
2141
1089
1 . . . 1089


Aurpu2p4_005861
2142
1571
1 . . . 279, 330 . . . 1571


Aurpu2p4_005992
2143
1362
1 . . . 1263, 1318 . . . 1362


Aurpu2p4_006091
2144
1466
1 . . . 246, 303 . . . 1466


Aurpu2p4_006277
2145
1656
1 . . . 219, 274 . . . 1656


Aurpu2p4_007520
2146
1230
1 . . . 115, 182 . . . 1001, 1065 . . . 1230


Aurpu2p4_007546
2147
578
1 . . . 115, 166 . . . 578


Aurpu2p4_007951
2148
2030
1 . . . 129, 185 . . . 205, 259 . . . 318, 382 . . . 722, 772 . . . 1063, 1116 . . . 2030


Aurpu2p4_008628
2149
1632
1 . . . 1632


Aurpu2p4_008719
2150
1026
1 . . . 124, 181 . . . 373, 440 . . . 759, 812 . . . 1026


Aurpu2p4_009254
2151
1936
1 . . . 212, 264 . . . 1264, 1329 . . . 1936


Aurpu2p4_009278
2152
1557
1 . . . 1557


Aurpu2p4_009437
2153
2129
1 . . . 47, 100 . . . 838, 891 . . . 2129


Aurpu2p4_009445
2154
1745
1 . . . 225, 278 . . . 559, 693 . . . 1745


Aurpu2p4_010136
2155
1764
1 . . . 110, 165 . . . 1764


Aurpu2p4_010244
2156
1860
1 . . . 1099, 1154 . . . 1310, 1368 . . . 1594, 1652 . . . 1860


Aurpu2p4_010617
2157
1041
1 . . . 1041


Aurpu2p4_010719
2158
1102
1 . . . 52, 111 . . . 563, 622 . . . 787, 991 . . . 1102


Aurpu2p4_010798
2159
1726
1 . . . 240, 290 . . . 634, 683 . . . 1726


Aurpu2p4_010869
2160
410
1 . . . 166, 228 . . . 313, 372 . . . 410









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


EXAMPLES
Example 1
Fermentation of the Organism
Materials & Methods

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.


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 (HCl) and bring volume to 1 L with double-distilled water.


Trace Element Solution contains 2 mM Iron(II) sulphate heptahydrate (FeSO4.7H2O), 1 mM Copper (II) sulphate pentahydrate (CuSO4.5H2O), 5 mM Zinc sulphate heptahydrate (ZnSO4.7H2O), 10 mM Manganese sulphate monohydrate (MnSO4.H2O), 5 mM Cobalt(II) chloride hexahydrate (CoCl2.6H2O), 0.5 mM Ammonium molybdate tetrahydrate ((NH4)6Mo7O24.4H2O), and 95 mM Hydrochloric acid (HCl) 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 (MgSO4.7H2O), 15 mg Calcium chloride dihydrate (CaCl2.2H2O), 100 μg 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








Varia-



tion
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.


TDM-4
The quantity of manganese sulphate monohydrate was raised



to 0.2 mM final concentration in the medium.


TDM-5
The quantity of copper (II) sulphate pentahydrate was



raised to 20 μM.


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 bran1.


TDM-9
Glucose was replaced with 10 g per liter of citrus pectin



(Sigma Cat. # P-9135).


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


TDM-11
The double-distilled water was replaced with whitewater2



collected from peroxide bleaching (which occurs during



the manufacture of fine paper).


TDM-12
The double-distilled water was replaced with whitewater2



collected from newsprint manufacture.


TDM-13
Glucose was replaced with 5 g per liter of ground



hardwood kraft pulp3.


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.


TDM-16
The strain was incubated at 10° C. below its optimum



growth temperature.


TDM-17
One half of the double-distilled water was replaced with



whitewater from newsprint manufacture. Glucose was



omitted.


TDM-18
Potassium phosphate monobasic was replaced with 5 mM



phytic acid from rice (Sigma Cat. # P3168).


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


TDM-20
Asparagine monohydrate was increased to 4 g per liter



and glucose was replaced with 2% fructose.


TDM-21
Asparagine monohydrate was increased to 4 g per liter;



100 mL of double-distilled water was replaced



with 100 mL kerosene4. Glucose was omitted.


TDM-22
Asparagine monohydrate was increased to 4 g per liter;



100 mL of double-distilled water was replaced with 100 mL



hexadecane (Sigma cat. # H0255). Glucose was omitted.


TDM-23
Asparagine monohydrate was increased to 4 g per liter;



one half of the double-distilled water was replaced with



25% whitewater from newsprint manufacture plus 25% white



water from peroxide bleaching. Glucose was omitted.


TDM-24
Asparagine monohydrate was increased to 4 g per liter



and the quantity of manganese sulphate monohydrate was



raised to 0.2 mM final concentration in the medium.


TDM-25
Asparagine monohydrate was increased to 4 g per liter



and manganese sulphate monohydrate was omitted from the



medium.


TDM-26
Asparagine monohydrate was increased to 4 g per liter;



and potassium phosphate monobasic was replaced with 5 mM



phytic acid from rice (Sigma Cat. # P3168).


TDM-27
Glucose was replaced with 10 g per liter of olive oil



(Sigma cat. # O1514)


TDM-28
One half of the double-distilled water was replaced with



whitewater from peroxide bleaching. 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).


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


TDM-33
Glucose was replaced with 20 g per liter of glycerol and



20 ml 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 10 g per liter of locust bean



gum (Sigma cat # G0753).


TDM-39
One half of double-distilled water was replaced with a



1:1 ratio of whitewater from newsprint manufacture and



white water from peroxide bleaching. Glucose was



omitted.


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


TDM-41
One half of double-distilled water was replaced with



whitewater from peroxide bleaching; plus yeast extract



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


TDM-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 20 g per liter of fructose.


TDM-44
Glucose was replaced with 10 g per liter of cellulose



(Solka-Floc, 200FCC) plus 1 g per liter of sophorose.


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






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




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




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




4Kerosene 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 SO4.7H2O, 1 mg Thiamine.









TABLE 4







Variations of AS media used for library construction








Varia-



tion
Description





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


AS-2
Glucose was replaced with 10 g per liter of pectin.


AS-3
One half of double-distilled water was replaced with a



1:1 ratio of whitewater from newsprint manufacture and



white water from peroxide bleaching. Glucose was omitted.









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).


The Scytalidium thermophilum, Myriococcum thermophilum, and Aureobasidium pullulans strains were each 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.


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

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

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.


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 μg/mL of DEPC-treated water.

    • 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:









(SEQ ID NO: 2935)


5′-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTT





TTTVN-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 by 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.


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 3730×1 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

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/InterProScan/) to assist in the identification of target enzymes.


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

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, and WO/2012/130964 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.


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.

  • Acetylxylan esterase CE5. 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; or Yang et al., International Journal of Molecular Sciences (2010), 11(12): 5143-5151.
  • Adhesin protein Mad1. 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).
  • Aldose 1-epimerase (mutarotase, aldose mutarotase). Polypeptides of the present invention having this activity can be characterized 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.
  • Allergen Asp f 15. Polypeptides of the present invention having this activity can be characterized 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 by Poutanen et al. (Appl. 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 U.S. Pat. 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-α-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. Bacteriol. (2002), 184: 4925-4929.
  • 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.
  • 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 Biomedicine 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 U.S. Pat. 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 GH79. 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-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 Fliedrová 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 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; U.S. Pat. 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.
  • 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.
  • 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 U.S. Pat. No. 7,087,810.
  • 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 U.S. Pat. No. 6,057,142.
  • 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 Cátia 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-Glo™ 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.
  • 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.
  • 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 U.S. Pat. No. 8,063,267.
  • Endoglycoceramidase. Polypeptides of the present invention having this activity can be characterized for example as described in U.S. Pat. No. 5,795,765; or US patent application publication No. US 2009/0170155 A1.
  • 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 A1; 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., Appl Environ Microbiol. (2006), 72(5): 3515-3523.
  • 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., Appl Microbiol Biotechnol. (2011), 89(3): 685-96; or Santos et al., J Bacteria (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, Appl Environ Microbiol. (2001), 67(7): 3319-3321.
  • 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., Journal 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-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 U.S. Pat. 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 U.S. Pat. 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.
  • 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 Ichinose et al., Applied and Environmental Microbiology (2008), 74(8): 2379-2383.
  • Gamma-glutamyltranspeptidase 2. Polypeptides of the present invention having this activity can be characterized for example as described in Rossi et al., PLoS One (2012), 7(2): e30543.
  • 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.
  • Glycosidase. Polypeptides of the present invention having this activity can be characterized for example as described in U.S. Pat. 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.
  • 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.
  • 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 U.S. Pat. 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 U.S. Pat. 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 U.S. Pat. Nos. 7,662,602 and 7,893,232.
  • 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 U.S. Pat. No. 5,965,422.
  • 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 Bacteria (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.
  • 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 Pleszczyńska, 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: February 22, PMID: 23436287; or in US patent application publication No. US20120258089 A1.
  • 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.
  • 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/Invitrogen); Cytochrome C Oxidase Assay Kit (Cat. No. CYTOCOX1-1KT; Sigma-Aldrich); Xanthine Oxidase Assay Kit (ab102522, Abcam); Lysyl Oxidase Activity Assay Kit (ab112139, Abcam); Glucose Oxidase Assay Kit (ab138884, Abcam); Monoamine oxidase B (MAOB) Specific Activity Assay Kit (ab109912, Abcam)].
  • 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 U.S. Pat. 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/Invitrogen); Peroxidase Activity Assay Kit (Cat. No. K772-100; BioVision); QuantiChrom™ Peroxidase Assay Kit (Cat. No. DPOD-100, BioAssay Systems].
  • 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/Invitrogen).
  • Polysaccharide monooxygenase. Polypeptides of the present invention having this activity can be characterized for example as described in Kittl 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. 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.
  • 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.
  • Rhamnogalacturonan lyase PL4. 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.
  • 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.
  • Serine-type carboxypeptidase F. Polypeptides of the present invention having this activity can be characterized for example as described in U.S. Pat. No. 6,379,913.
  • Swollenin. Polypeptides of the present invention having this activity can be characterized for example as described in Jäger et al., Biotechnol Biofuels. (2011), 4: 33; or Saloheimo et al., Eur J Biochem. (2002), 269(17): 4202-11.
  • 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.
  • 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.
  • 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; U.S. Pat. No. 7,759,102; or PCT application publication No. WO 2006/078256 A2.
  • Xyloglucanase GH12. 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.
  • 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 U.S. Pat. No. 6,077,702; Damásio 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

Standard molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, E. 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, N.Y. 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 3730×1 DNA Analyzer technology at the Innovation Centre (Génome Québec), McGill University in Montreal.


Example 7
Construction of pGBFIN49 Expression Plasmids

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 TrpC terminator, A. nidulans gpdA promoter, gene encoding the pheomycin resistance gene, A. niger glaA terminator and an E. coli backbone. FIG. 1 represents a schematic map of pGBFIN-49 and the complete nucleotide sequence is presented as SEQ ID NO: 2936. Details of the construction of pGBFIN-49 are as follows:


1. TtrpC Terminator PCR Amplification (0.7 kb):

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









(SEQ ID NO: 2937)


Primer-3: 5′-GTCCGTCGCCGTCCTTCAccgccggtccgacg-3′





(SEQ ID NO: 2938)


Primer-4: 5′-GCGGCCGGCGTATTGGGTGttacggagc-3′






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
μL (5-10 ng)



Primer-3 (10 mM)
1
μL



Primer-4 (10 mM)
1
μL



dNTPs (2 mM)
5
μL



HF Buffer (5x)
10
μL



Phusion DNS pol.
0.5
μL



Nuclease-free water
31.5
μL



Total
50
μL










PCR Program:


1×98° C., 2 min; 25×(98° C., 30 sec; 68° C., 30 sec; 72° C., 1 min); 72° C., 7 min.


Reaction conditions: 5 μL 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 (8.3 kb):


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:









(SEQ ID NO: 2939)


Primer-2: 5′-CACCCAATACGCCGGCCGCgcttccagacagctc-3′





(SEQ ID NO: 2940)


Primer-1C: 5′-GGTGTTTTGTTGCTGGGGAtgaagctcaggctctca


gttgcgtc-3′






Primer-2 contains a pgpdA-specific region and an extra sequence specific to TtrpC 3′ end (also included in Primer-4). Primer-1C 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
μL (50 ng)



Primer-2 (10 mM)
1
μL



Primer-1C (10 mM)
1
μL



dNTPs (2 mM)
5
μL



HF Buffer (5x)
10
μL



Phusion DNS pol.
0.5
μL



DMSO
1
μL



Nuclease-free water
30.5
μL



Total
50
μL










PCR Program:


1×98° C., 3 min; 10×(98° C., 30 sec; 68° C., 30 sec, 72° C., 5 min); 20×(98° C., 30 sec, 68° C., 30 sec, 72° C., 5 min+10 sec/cycle); 72° C., 10 min.


Reaction Conditions:


5 μL 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:


Overlap-extension/Long range PCR was performed to: a) fuse the two PCR pieces together; b) add an SfoI 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.











(SEQ ID NO: 78)



Primer-11: 5′-CACCGGCGCCGTCCGTCGCCGTCCTTC-3′






(SEQ ID NO: 79)



Primer-12: 5′-ACGGCGCCGGTGTTTTGTTGCTGGGGATG-3′






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 SfoI restriction site sequence is underlined above.


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 μL



pGBFIN41
9 μL



Buffer GC (5x)
10 μL 



dNTPs (2 mM)
5 μL



Phusion DNA pol.
0.5 μL  



Nuclase-free water
24.5 μL  



pGBFIN41
50 μL 










PCR Program—Overlap (No Primers):


1×98° C., 2 min; 5×(98° C., 15 sec; 58° C., 30 sec; 72° C., 5 min), 5×(98° C., 15 sec; 63° C., 30 sec; 72° C., 5 min), 5×(98° C., 15 sec; 68° C., 30 sec; 72° C., 5 min); 72° C., 10 min.


The overlap-extension PCR product was then, purified on QIAEX II™ column and 5 μL 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 μL



Primer-11 (10 mM)
1 μL



Primer-12 (10 mM)
1 μL



dNTPs (2 mM)
5 μL



HF Buffer (5x)
10 μL 



Phusion DNA pol.
0.5 μL  



DMSO
1 μL



Nuclease-free water
26.5 μL  



pGBFIN41
50 μL 










PCR Program—Long Range:


1×98° C., 3 min; 10×(98° C., 30 sec; 68° C., 30 sec; 72° C., 5 min); 20×(98° C., 30 sec; 68° C., 30 sec; 72° C., 5 min+10 sec/cycle); 72° C., 10 min.


Reaction Conditions:


5 μL 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, SfoI digestion was performed and digested product was purified using QIAEX II gel extraction kit follow the procedure as described by the manufacturer.


4. Ligation:

100 ng of the purified digested fragment was ligated to itself using 1 μL 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 μL 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 Scytalidium thermophilum, Myriococcum thermophilum, and Aureobasidium pullulans genes in E. coli

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.


Coding sequences from genes of interest were amplified by PCR using primers containing LIC tags, which are homologous to Pgla 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:









(SEQ ID NO: 2941)


Forward primer: 5′-CCCCAGCAACAAAACACCTCAGCAATG...


15-20 nucleotides specific to each gene to be


cloned





(SEQ ID NO: 2942)


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/μL
  1 μL



5X Phusion HF Buffer (Finnzymes ™)
 10 μL



2 mM dNTPs
  5 μL



LIC primer (F + R) mix 10 mM
0.5 μL



Phusion DNA Polymerase (Finnzymes ™)
0.5 μL



DMSO
1.5 μL



H2O
31.5 μL 



TOTAL
 50 μL










PCR Amplification was Carried Out with Following Conditions:
















3-step protocol













Cycle step
Temp
Time
Cycles

















Initial denaturation
98° C.
30
s
1



Denaturation
98° C.
10
s
10



Annealing
58° C.
30
s



Extension
72° C.
30
s



Denaturation
98° C.
10
s
20



Annealing
68° C.
30
s



Extension
72° C.
30
s



Final extension
70° C.
10
min
1












End of PCR storage
 4° C.
hold
1










Following PCR, 90 μL milliQ™ water was added to each sample and the mix was purified using a MultiScreen PCR96 Filter Plate (Millipore) according to manufacturer's instructions. The PCR product was eluted from the filter in 25 μL 10 mM Tris-HCl pH 8.0.


Expression Vector pGBFIN-49 was PCR Amplified Using Primers with Following Sequences:









(SEQ ID NO: 2943)


Forward primer: 5′-GTCCGTCGCCGTCCTTCACCG-3′





(SEQ ID NO: 2944)


Reverse primer: 5′-GGTGTTTTGTTGCTGGGGATGAAGC-3′






Primers are Located at Either Site of the SfoI Restriction Site

PCR Mix Consists of Following Components:


















pGBFIN-49 plasmid DNA (10 ng/μL)
 2 μL



5X Phusion HF Buffer (Finnzymes ™)
20 μL



2 mM dNTPs
10 μL



LIC Primer mix (F + R) 10 mM
 2 μL



Phusion DNA Polymerase (Finnzymes ™)
1.5 μL 



DMSO
 3 μL



H2O
61.5 μL  



TOTAL
100 μL 










PCR Amplification was Carried Out with Following Conditions:
















2-step PCR protocol











Cycle step
Temp.
Time
Cycles













Initial denaturation
98° C.
 2 min
1


Denaturation
98° C.
10 s
35


Annealing + Extension
68° C.
4 min + 10 s/cycle


Final extension
70° C.
10 min
1


End of PCR storage
 4° C.
Hold
1









Following PCR, 1 μL of DpnI 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.


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.


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
μL



25 mM dTTP
2
μL



DTT 100 μM
0.8
μL



T4 DNA Polymerase 3 U/μL
1
μL



H2O
Up to 20
μL



TOTAL
20
μL










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 μL



10X NEB Buffer 2
2 μL



25 mM dATP
2 μL



DTT 100 μM
0.8 μL  



T4 DNA Polymerase 3 U/μL
1 μL



H2O
9.2 μL  



TOTAL
20 μL 










Reaction Conditions were as Follows:

















Step
Temperature (° C.)
Duration




















Annealing
22
30 min



Enzyme inactivation
75
20 min



End
4
Hold










Following T4 DNA polymerase treatment, 2 μL of pGBFIN-49 vector and 4 μL 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 Scytalidium thermophilum, Myriococcum Thermophilum, and Aureobasidium pullulans Gene Expression Cassettes into A. niger

As host strain for enzyme production, A. niger GBA307 was used. Construction of A. niger GBA307 is described in WO 2011/009700.


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 J., 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 NaNO3; 0.52 g KCl; 1.52 g KH2PO4; 1.12 ml 4 M KOH; 0.52 g MgSO4.7H2O; 10 g glucose; 1 g casamino acids; 22 mg ZnSO4.7H2O; 11 mg H3BO3; 5 mg FeSO4.7H2O; 1.7 mg CoCl2.6H2O; 1.6 mg CuSO4.5H2O; 5 mg MnCl2.2H2O; 1.5 mg Na2MoO4.2H2O; 50 mg EDTA; 2 mg riboflavin; 2 mg thiamine-HCl; 2 mg nicotinamide; 1 mg pyridoxine-HCl; 0.2 mg panthotenic acid; 4 μg biotin; 10 ml Penicillin (50001 U/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-HCl pH 7.5) was pipetted gently on top of the protoplast suspension. After centrifugation for 10 min at 4330×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-HCl pH 7.5, 50 mM CaCl2). The protoplast suspension was centrifuged for 10 min at 1560×g in a swinging bucket rotor and resuspended in STC-buffer at a concentration of 108 protoplasts/mL;
    • To 200 μL of the protoplast suspension, 20 μL ATA (0.4 M Aurintricarboxylic acid), the DNA dissolved in 10 μL in TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA), 100 μL of a PEG solution (20% PEG 4000 (Merck), 0.8M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl2) 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-HCl pH7.5, 50 mM CaCl2) 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×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.HCl, nicotinamide, pyridoxine, panthotenic acid, biotin, casamino acids and glucose, supplemented with 150 μg/mL Phleomycin (Invitrogen), 0.07 M NaNO3, 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 μg/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

96 wells microtiter plates (MTP) with sporulated Aspergillus niger strains were used to harvest spores for MTP fermentations. To do this, 100 ?l water was added to each well and after resuspending the mixture, 40 μL of spore suspension was used to inoculate 2 mL A. niger medium (70 g/L glucose.H2O, 10 g/L yeast extract, 10 g/L (NH4)2SO4, 2 g/L K2SO4, 2 g/L KH2PO4, 0.5 g/L MgSO4.7H2O, 0.5 g/L ZnSO4.7H2O, 0.2 g/L CaCl2, 0.01 g/L MnSO4.7H2O, 0.05 g/L FeSO4.7H2O, 0.002 Na2MoO4.2H2O, 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 μM CuSO4 was included in the media. The MTP's were incubated in a humidity shaker (Infors) 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

Approximately 1×108-1×107 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; KH2PO4 1 g/L; MgSO4.7H2O 0.5 g/L; ZnCl2 0.03 g/L; CaCl2 0.02 g/L; MnSO4.4H2O 0.01 g/L; FeSO4.7H2O 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.H2O 70 g/L; Peptone (aus casein) 25 g/L; Yeast extract 12.5 g/L; K2SO4 2 g/L; KH2PO4 1 g/L; MgSO4.7H2O 0.5 g/L; ZnCl2 0.03 g/L; CaCl2 0.02 g/L; MnSO4.1H2O 0.009 g/L; FeSO4.7H2O 0.003 g/L; pH 5.6.


Note: for GH61 (e.g., polysaccharide monooxygenase) enzymes the culture media were supplemented with 10 μM CuSO4.


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×g followed by germ-free filtration of the supernatant over 0.2 μm PES filters (Nalgene).


Example 12
Protein Concentration Determination with TCA-Biuret Method

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
Microtiter Plate (MTP) Sugar-Release Activity Assay

For each (hemi-)cellulase assay, the stored samples were analyzed twice according the following procedure 100 μL sample and 100 μL of a (hemi-)cellulase base mix [1.75 mg/g DM TEC-210 or a 3 enzyme mix at a total dosage of 3.5 mg/g DM consisting of 0.5 mg/g DM BG (14% of total protein 3E mix), 1.6 mg/g DM CBHI (47% of total protein 3E mix) and 1.4 mg/g DM CBHII (39% of total protein 3E mix)] was transferred to two suitable vials: one vial containing 800 μL 2.5% (w/w) dry matter of the acid pre-treated corn stover substrate in a 50 mM citrate buffer, buffered at pH 4.5. The other vial consisted of a blank, where the 800 μL 2.5% (w/w) dry matter, acid pre-treated corn stover substrate suspension was replaced by 800 μL 50 mM citrate buffer, buffered at pH 4.5. The assay samples were incubated for 72 hrs at 65° C. After incubation of the assay samples, a fixed volume of an internal standard, maleic acid (20 g/L), EDTA (40 g/L) and DSS (0.5 g/L), was added. After centrifugation, the supernatant of the samples is lyophilized overnight; subsequently 100 μL D2O is added to the dried residue and lyophilized once more. The dried residue is dissolved in 600 μL of D2O.


The amount of sugar released, is based on the signal between 4.65-4.61 ppm, relative to DSS, and is determined by means of 1D 1H NMR operating at a proton frequency of 500 MHz, using a pulseprogram without water suppression, at a temperature of 27° C.


The (hemi)-cellulase enzyme solution may contain residual sugars. Therefore, the results of the assay are corrected for the sugar content measured after incubation of the enzyme solution.


Example 14
Sugar-Release Activity Assays: Labscale, Incubation with Shaking


A. niger strains expressing Scytalidium thermophilum, Myriococcum thermophilum, and Aureobasidium pullulans clones were grown in shake flask, as described above (Example 11), in order to obtain greater amounts of material for further testing. The fermentation supernatants (volume between 40 and 80 mL) were concentrated using a 10-kDa spin filter to a volume of approximately 5 mL. Subsequently, the protein concentration in the concentrated supernatant was determined via a TCA-biuret method, as described above in Example 12. The (hemi-)cellulase activity of these protein samples was tested in an assay where the supernatants were spiked on top of an enzyme base mix in the presence of 10% (w/w) acid pretreated corn stover (aCS). ‘To spike’ or ‘spiking of’ a supernatant or an enzyme indicates, in this context, the addition of a supernatant or an enzyme to a (hemi)-cellulase base mix. The feedstock solution was prepared via the dilution of a concentrated feedstock solution with water. Subsequently, the pH was adjusted to pH 4.5 with a 4 M NaOH solution. The proteins were spiked based on dosage; the concentrated supernatant samples were added in a final concentration of 2 mg/gDM to the base enzyme mix (TEC-210 5 mg/gDM) in a total volume of 10 mL at a feedstock concentration of 10% aCS (w/w) in an 30-mL centrifuge bottle (Nalgene Oakridge). All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described below.


Example 15
Soluble Sugar Analysis by HPLC

The sugar content of the samples after enzymatic hydrolysis were analyzed using a High-Performance Liquid Chromatography System (Agilent 1100) equipped with a refection index detector (Agilent 1260 Infinity). The separation of the sugars was achieved by using a 300×7.8 mm Aminex HPX-87P (Bio-Rad cat. no. 125-0098) column; Pre-column: Micro guard Carbo-P (Bio-Rad cat. no. 125-0119); mobile phase was HPLC grade water; flow rate of 0.6 mL/min and a column temperature of 85° C. The injection volume was 10 μL.


The samples were diluted with HPLC grade water to a maximum of 10 g/L glucose and filtered by using 0.2 μm filter (Afridisc LC25 mm syringe filter PVDF membrane). The glucose was identified and quantified according to the retention time, which was compared to the external glucose standard (D-(+)-Glucose Sigma cat. no: G7528) ranging from 0.2; 0.4; 1.0; 2.0 g/L.


Example 16
Protein Activity Assays

16.1 Alpha-Arabino(Furano)Sidase Activity Assay


This assay measures the ability of α-arabino(furano)sidases to remove the alpha-L-arabinofuranosyl residues from substituted xylose residues. Single and double substituted oligosaccharides are prepared by incubating wheat arabinoxylan (WAX medium viscosity; 2 mg/mL; Megazyme, Bray, Ireland) in 50 mM acetate buffer pH 4.5 with an appropriate amount of endo-xylanase (Aspergillus Awamori, F J M, Kormelink, Carbohydrate Research, 249 (1993) 355-367) for 48 hours at 50° C. to produce an sufficient amount of arabinoxylo-oligosaccharides. The reaction is stopped by heating the samples at 100° C. for 10 minutes. The samples are centrifuged for 5 minutes at 10,000×g. The supernatant is used for further experiments. Degradation of the arabinoxylan is followed by High Performance Anion Exchange Chromatography (HPAEC).


The enzyme is added to the single and double substituted arabinoxylo-oligosaccharides (endo-xylanase treated WAX) in a dosage of 10 mg protein/g substrate in 50 mM sodium acetate buffer which is then incubated at 65° C. for 24 hours. The reaction is stopped by heating the samples at 100° C. for 10 minutes. The samples are centrifuged for 5 minutes at 10,000×g and 10 times diluted. Release of arabinose from the arabinoxylo-oligosaccharides is analyzed by HPAEC analysis.


The analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with a CarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM. Each elution is followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH. Arabinose release is quantified by an arabinose standard (Sigma) and compared to a sample where no enzyme was added.


16.2 Beta-Xylosidase Activity Assay

This assay measures the release of xylose by the action of beta-xylosidase on xylobiose. Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows. 4.1 g of anhydrous sodium acetate or 6.8 g of sodium acetate*3H2O is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is equal to 4.5.


Xylobiose was purchased from Sigma and a solution of 100 μg/mL sodium acetate buffer pH 4.5 was prepared. The assay is performed as detailed below.


The enzyme is added to the substrate in a dosage of 10, 5 or 1 mg protein/g substrate, which is then incubated at 62-65° C. for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100° C. Samples are appropriate diluted and the release of xylose is analyzed by High Performance Anion Exchange Chromatography.


The analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with a CarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-20 min, 0-17.8 mM. Each elution is followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.


In case interfering compounds are present that complicate xylose quantification, the analysis is performed by running isocratic on H2O for 30 min a gradient (0.5M NaOH is added post-column at 0.1 mL/min for detection) followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min H2O.


Standards of xylose and xylobiose (Sigma) are used for identification and quantification of the substrate and product formed by the enzyme.


16.3 Acetyl-Xylan Esterase Activity Assay

Acetyl-xylan esterases are enzymes able to hydrolyze ester linked acetyl groups attached to the xylan backbone, releasing acetic acid. This assay measures the release of acetic acid by the action of acetyl xylan esterase on acid pretreated corn stover (aCS) that contains ester linked acetyl groups.


Determine the Presence of Acetyl Groups in pCS

The aCS used contains ±284 (±5.5) μg acetic acid/20 mg pCS as determined according to the following method.


About 20 mg of aCS substrate was weighed in a 2 mL reaction tube and placed in an ice-water bath. Then 1 mL of 0.4M NaOH in Millipore water/isopropanol (1:1) was added and the sample was thoroughly mixed. This was incubated on ice for 1 hour. Subsequently, the samples were mixed again and incubated for 2 additional hours at room temperature (mixed once in a while). After this samples were centrifuged for 5 min at 12000 rpm and the supernatant was analyzed for acetic acid content by HPLC.


Enzyme Incubations

Enzyme incubations were performed in citrate buffer (0.05 M, pH 4.5) which is prepared as follows; 14.7 g of tri-sodium citrate is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 10.5 g citric acid monohydrate is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium citrate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.


The aCS substrate is solved in citrate buffer to obtain ±20 mg/mL. The enzyme is added to the substrate in a dosage of 1 or 10 mg protein/g substrate, which is then incubated at 60° C. for 24 hours head-over-tail. The reaction is stopped by heating the samples for 10 minutes at 100° C. The release of acetic acid is analyzed by HPLC.


As a blank sample the substrate is treated and incubated in the same way but then without the addition of enzyme.


The analysis is performed using an Ultimate 3000 system (Dionex) equipped with a Shodex RI detector and an Aminex HPX 87H column (7.8 mm ID×300 mm) column (BioRad). A flow rate of 0.6 mL/min is used with 5.0 mM H2SO4 as eluent for 30 minutes at a column temperature of 40° C. Acetic acid was used as a standard to quantify its release from pCS by the enzymes.


16.4 Endoxylanase Activity Assay 1

Endoxylanases are enzyme able to hydrolyze β-1,4 bond in the xylan backbone, producing short xylooligosaccharides. This assay measures the release of xylose and xylo-oligosaccharides by the action of xylanases on wheat arabinoxylan oligosaccharides (WAX) (Megazyme, Medium viscosity 29 cSt) and Beech Wood Xylan (Beech) (Sigma).


Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows; 4.1 g of anhydrous sodium acetate is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.


The substrates WAX and Beech are solved in sodium acetate buffer to obtain 2.0 mg/mL. The enzyme is added to the substrate in a dosage of 10 mg protein/g substrate which is then incubated at 65° C. for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100° C. The release of xylose and (arabino)xylan oligosaccharides is analyzed by High Performance Anion Exchange Chromatography.


As a blank sample the substrate is treated and incubated in the same way but then without the addition of enzyme.


The analysis is performed using a Dionex HPLC system equipped with a Dionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with a CarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector (Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min is used with the following gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM. Each elution is followed by a washing step of 5 min 1000 mM sodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH. Standards of xylose, xylobiose, xylotriose and xylotetraose (Sigma) are used to identify and quantify these oligomers released by the action of the enzyme.


16.5 Endo-Xylanase Activity Assay 2

Endo-xylanases are enzyme able to hydrolyze beta-1,4 bond in the xylan backbone, producing short xylooligosaccharides. This assay measures the release of xylose and xylo-oligosaccharides by the action of xylanases on wheat arabinoxylan oligosaccharides (WAX) (Megazyme, Medium viscosity 29 cSt).


Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows: 4.1 g of anhydrous sodium acetate is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.


The substrate WAX is solved in sodium acetate buffer to obtain 2.0 mg/mL. The enzyme is added to the substrate in a dosage of 1 mg protein/g substrate which is then incubated at 65° C. for 24 hours. During these 24 hours, samples are taken and the reaction is stopped by heating the samples for 10 minutes at 100° C.


The enzyme activity is demonstrated by using a reducing sugars assay (PAHBAH) as detection method.


Reagent A: 5 g of p-Hydroxybenzoic acid hydrazide (PAHBAH) is suspended in 60 mL water, 4.1 mL of concentrated hydrochloric acid is added and the volume is adjusted to 100 mL. Reagent B: 0.5 M sodium hydroxide. Both reagents are stored at room temperature. Working Reagent: 10 mL of Reagent A is added to 40 mL of Reagent B. This solution is prepared freshly every day, and is stored on ice between uses. Using the above reagents, the assay is performed as detailed below.


The assay is conducted in microtiter plate format. After incubation 10 μL of each sample is added to a well and mixed with 150 μL working reagent. These solutions are heated at 70° C. for 30 minutes or for 5 minutes at 90° C. After cooling down, the samples are analyzed by measuring the absorbance at 405 nm. The standard curve is made by treating 10 μL of an appropriate diluted xylose solution the same way as the samples. The reducing-ends formed due to the action of enzyme is expressed as xylose equivalents.



Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands in December 1964 having the Accession Number CBS 393.64.


Other suitable strains can be equally used in the present examples to show the effect and advantages of the invention. For example TEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsonia strains which are described in WO 2011/000949. The “4E mix” or “4E composition” was used containing CBHI, CBHII, EG4 and BG (30 wt %, 25 wt %, 28 wt % and 8 wt %, respectively, as described in WO 2011/098577, wt % on dry matter protein).



Rasamsonia (Talaromyces) emersonii strain TEC-101 (also designated as FBG 101) was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 30, Jun. 2010 having the Accession Number CBS 127450.


TEC-210 was fermented according to the inoculation and fermentation procedures described in WO 2011/000949.


The 4E mix (4 enzymes mixture or 4 enzyme mix) containing CBHI, CBHII, GH61 and BG (30%, 25%, 36% and 9%, respectively as described in WO 2011/098577) was used.


3E mix (3 enzymes mixture or 3 enzyme mix) is spiked with a fourth enzyme to form the 4E mix.


16.6 Xyloglucanase Activity Assay

Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows: 4.1 g of anhydrous sodium acetate is dissolved in distilled water to a final volume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) of glacial acetic acid is mixed with distilled water to make the total volume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer, pH 4.5, is prepared by mixing Solution A with Solution B until the pH of the resulting solution is 4.5.


Tamarind xyloglucan is dissolved in sodium acetate buffer to obtain 2.0 mg/mL. The enzyme is added to the substrate in a dosage of 10 mg protein/g substrate, which is then incubated at 60° C. for 24 hours. The reaction is stopped by heating the samples for 10 minutes at 100° C. The formation of lower molecular weight oligosaccharides is analyzed by High Performance size-exclusion Chromatography


As a blank sample, the substrate is treated and incubated in the same way but then without the addition of enzyme.


The analysis is performed using High-performance size-exclusion chromatography (HPSEC) performed on three TSK-gel columns (6.0 mm×15.0 cm per column) in series SuperAW4000, SuperAW3000, SuperAW2500; Tosoh Bioscience), in combination with a PWXguard column (Tosoh Bioscience). Elution is performed at 55° C. with 0.2 M sodium nitrate at 0.6 mL/min. The eluate was monitored using a Shodex RI-101 (Kawasaki) refractive index (RI) detector. Calibration was performed by using pullulans (Associated Polymer Labs Inc., New York, USA) with a molecular weight in the range of 0.18-788 kDa.


16.7 Assay Protocol CU1: Colorimetric Assay for Glycosidase or Esterase Activity, Measuring Release of 4-Nitrophenol

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 μL of diluted enzyme sample is added to 30 μL 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 μL of preheated 5 mM substrate in water (see Table 5) to buffer and sample. Standards contain 10 μL 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 μL of resulting 100 mM solution to 10 mL in water) and 40 μL of reaction buffer. Sample blank contains 10 μL of enzyme sample and 40 μL of reaction buffer. Substrate blank contains 10 μL of substrate (see table) and 40 μL of reaction buffer. After appropriate incubation time, 50 μL of [1] for 4-nitrophenyl acetate, 1 M HEPES buffer pH 8 in water; [2] for 4-nitrophenyl butyrate, 250 mM Na2CO3 in water; [3] for all other substrates, 1 M Na2CO3 in water is added. 80 μL 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 5







Enzyme activity
Substrate









arabinofuranosidase
4-nitrophenyl alpha-L-arabinofuranoside



arabinopyranosidase
4-nitrophenyl alpha-L-arabinopyranoside



beta-galactosidase
4-nitrophenyl beta-D-galactopyranoside



hexosaminidase/N-
4-nitrophenyl beta-D-glucosaminide



acetylglucosaminidase



beta-glucosidase
4-nitrophenyl beta-D-glucopyranoside



beta-mannosidase
4-nitrophenyl beta-D-mannopyranoside



beta-xylosidase
4-nitrophenyl beta-D-xylopyranoside



Acetylesterase
4-nitrophenyl acetate



Cutinase; lipase
4-nitrophenyl butyrate










16.8 Assay Procedure CU2: Colorimetric Assay for Endo-Glycanase Activity, Measuring Copper (I) Reduced by Polysaccharide Reducing Ends

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 μL of diluted sample is added to 30 μL 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 μL of preheated 5 mM substrate in water (see Table 6) to buffer and sample. Standards contain 10 μL of 0 to 7.5 mM monosaccharide solution (see Table 6) in water and 40 μL of reaction buffer. Enzyme sample blank contains 10 μL of sample and 40 μL of reaction buffer. Substrate blank contains 10 μL of substrate (see Table 6) and 40 μL of reaction buffer. After appropriate incubation time, 10 μL is removed and added to another PCR plate containing 95 μL of BCA Reagent A (made by dissolving 0.543 g Na2CO3, 0.242 g NaHCO3 and 19 mg disodium 2,2′-bicinchoninate in water and diluting to 1 L) and 95 μL of BCA Reagent B (made by dissolving 12 mg CuSO4 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 μL 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.)











TABLE 6





Enzyme
Substrate
Standard







Tomatinase
alpha-tomatine
galactose


Endomannanase
Beta-Mannan
Mannose


Endoglucanase
Carboxymethyl
Glucose



cellulose (1:1 mixture



of 4M and 7M)


Laminarinase
Laminarin
Glucose


Lichenanase
Lichenan
Glucose


Endomannanase
Locust bean gum
Mannose


Arabinoxylan
Low Viscosity Wheat
Arabinose


arabinofuranohydrolase
Arabinoxylan


Endopolygalacturonase
Polygalacturonic acid
Galacturonic acid


Sucrase/Alpha-glucosidase/
Sucrose
Glucose + Fructose


Invertase

(1:1 mixture)









16.9 Assay Procedure CU3: UV Assay for Acetylesterase Activity, Measuring Release of Alpha-Naphthol

Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in dH2O, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L. 20 μL of diluted sample is added to 20 μL of 300 mM acetate-phosphate-borate reaction buffer at appropriate pH (made by dissolving 17.28 mL 99.7% glacial acetic acid, 20.52 mL 85% phosphoric acid, and 18.6 g boric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) in a clear microtiter plate and preheated to appropriate temperature in the plate reader. The reaction is started by addition of 160 μL 0.5 mM alpha-naphthyl acetate substrate solution in water (prepared by diluting 46.55 mg of a-Naphthyl acetate in 1 mL of acetone and then transferring to 499 mL of water), preheated to assay temperature in a dry block heater, to the buffer and enzyme sample. Standards contain 180 μL of 0 to 0.1 mM alpha-naphthol in water and 20 μL of reaction buffer. Blank contains 20 μL of reaction buffer, 20 μL of water and 160 μL of substrate solution. Absorbance is continuously monitored at 303 nm and compared to that of the standards. One unit is the amount of enzyme that produces one micromole of alpha-naphthol per minute under the specified conditions. (Adapted from Yuorno et al. (1981), Anal. Biochem. 115, 188-193)


16.10 Assay Procedure CU4: Polarimetric Assay for Aldose 1-Epimerase Activity, Measuring the Rate Increase of the Mutarotation of Alpha-D-Glucose

5 mM phosphate reaction buffer (prepared by dissolving 342 μL 85% phosphoric acid in water, adjusting to pH 5.0 with 1 M NaOH and diluting to 1 L) is preheated to 40° C. A Perkin-Elmer 341 polarimeter (USA) with sodium/halogen and mercury lamps preheated to 40° C. and is blanked by measuring the optical rotation of polarized 578 nm light by 5 mL reaction buffer. 36 mg of alpha-D-Glucose is dissolved in 10 mL of reaction buffer, then 60 μL of undiluted enzyme is added to 4.94 mL of the resulting solution and optical rotation is immediately measured in the polarimeter. Readings are recorded at 40° C. every minute until equilibrium is reached. One unit is the amount of enzyme that converts one micromole of alpha-D-glucose to beta-D-glucose (calculated by determining the reaction's first-order rate constant less that of the blank) in one minute. (Adapted from Bailey et al. (1975), Methods in Enzymology 41, 471-484).


16.11 Assay Procedure CU5: Colorimetric Assay Measuring Acid Release by the Absorbance of a pH Indicator

Reaction buffer is 2.5 mM MOPS, pH 7.2 (0.52 g MOPS dissolved in water, pH adjusted with 1 M NaOH and diluted to 1 L) or 2.5 mM acetate, pH 5.3 (144 μL glacial acetic acid dissolved in water, pH adjusted with 1 M NaOH and diluted to 1 L). Substrate stock solution is made by dissolving 111.1 mg of ethyl ferulate and 70 mg of 4-nitrophenol or 350 mg of bromocresol green in isopropyl alcohol. Substrate working solution is made by diluting substrate stock solution 1:10 with reaction buffer: pH 7.2 reaction buffer is used for substrate stock solution containing 4-nitrophenol, pH 5.3 for stock containing bromocresol green. Enzyme is thoroughly buffer exchanged into reaction buffer before use in the assay. Enzyme and substrate working solution are preheated to the appropriate temperature; 100 μL substrate working solution is added to a microtiter plate, and 20 μL of enzyme solution is added. The change in absorbance at 410 nm (pH 7.2) or 600 nm (pH 5.3) is determined. The pH of the solution is calculated by comparing the absorbance to that of the blank, and the amount of acid released is calculated. One unit is defined as the amount of enzyme that produces one micromole of ferulic acid per minute. (Adapted from Ramirez et al. (2008), Appl Biochem Biotechnol 151, 711-723.)


16.12 Assay Procedure CU6: UV Assay of Lyase Activity, Measuring Formation of Unsaturated Bonds

Enzyme sample is diluted in 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, 3.10 g boric acid and 1.11 g calcium chloride in water, adjusting pH with 10 M NaOH and diluting to 1 L) and left to equilibrate for 30 minutes at room temperature. Reaction buffer is mixed in a 1:1 ratio with substrate solution (1% polygalacturonic acid in water or 0.75% Rhamnogalacturonan I from potato in water) and preheated to reaction temperature in a dry bath heater (if reaction temperature is greater than plate reader maximum temperature) or in a microtiter plate in plate reader. Reaction is started by addition of 10 μL of diluted enzyme sample to 240 μL of reaction buffer/substrate in UV-transparent microtiter flat-bottomed plate. Blank contains 10 μL of reaction buffer added to 240 μL of reaction buffer/substrate solution. Absorbance at 235 nm is continuously monitored, and the molar absorptivity coefficient of unsaturated galacturonic acid is used to determine activity. One unit is the amount of enzyme that releases one micromole of unsaturated galacturonic acid equivalents per minute under the specified conditions. Adapted from Hansen et al. (2001) J. AOAC International, 84, 1851-1854)


16.13 Assay Procedure CU7: Fluorescence Assay, Measuring Release of 4-Methylumbelliferone

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 μL of diluted sample is added to 30 μL 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. The reaction is started by addition of 10 μL of preheated 1 mM substrate in water (made by diluting 5.0 mg of 4-methylumbelliferyl cellobioside or 4-methylumbelliferyl lactoside in 10 mL water) to buffer and sample. Standards contain 10 μL of 4-methylumbelliferone (from 0 to 50 uM; 19.8 mg of 4-methylumbelliferone sodium salt is dissolved in 100 mL methanol and resulting solution is diluted 20× in water) and 40 μL of reaction buffer. Enzyme sample blank contains 10 μL of enzyme sample and 40 μL of reaction buffer. Substrate blank contains 10 μL of substrate and 40 μL of reaction buffer. After appropriate incubation time, 20 μL is removed and added to a black microtiter plate containing 180 μL of glycine/carbonate buffer, pH 10.7 (made by dissolving 10 g glycine and 8.8 g sodium carbonate in water, adjusting pH with 10 M NaOH and diluting to 1 L). The fluorescence of the wells is measured at 355 nm excitation, 460 nm emission and compared to the standard curve. One unit is defined as the amount of enzyme that releases one micromole of 4-methylumbelliferone per minute. (Adapted from van Tilbeurgh et al. (1988), Methods in Enzymology 160: 45-59.)


16.14 Assay Procedure CUB: Spectrophotometric Assay of Acetylxylanesterase Activity, Measuring Release of Acetic Acid

Enzyme sample is diluted in 10 mM citrate buffer, pH 5.0, made by dissolving 1.92 g of citric acid in dH2O, adjusting pH to 5.0 with 10 M NaOH and diluting to 1 L. 40 μL of 1% acetylated xylan from birchwood are added to 40 μL of 50 mM phosphate reaction buffer (prepared by dissolving 3.42 mL of 85& phosphoric acid in water, adjusting pH to 6.0 with 10 M NaOH and diluting to 1 L) in the wells of a 96-well PCR plate and preheated to the appropriate temperature in a dry block heater. The reaction is started by adding 20 μL of diluted sample to the wells containing substrate and reaction buffer. Standards contain 20 μL of 0 mg/mL to 1 mg/mL acetic acid in water, and 80 ul reaction buffer. Sample blank contains 20 μL of diluted enzyme sample, 40 μL of reaction buffer and 40 μL of water. Substrate blank contains 40 μL of substrate and 60 μL of reaction buffer. After appropriate incubation time, the plate is heated to 90° C. for 5 minutes and centrifuged 10 minutes at 1500×g. The amount of acetic acid in the supernatant is then determined with the K-ACETAK kit by Megazyme; one unit is defined as the amount of enzyme required to release one micromole of acetic acid per minute under the specified conditions. (Adapted from Johnson et al. (1988), Methods in Enzymology 160, 551-560 and K-ACETAK assay kit procedure by Megazyme (Ireland)).


16.15 Assay Procedure CU9: Gas Chromatographic Assay of Methylesterase Activity, Measuring Release of Methanol

Reaction buffer is 50 mM phosphate, pH 6.6, made by dissolving 3.42 mL 85% phosphoric acid in water, adjusting pH with 10 M NaOH and diluting to 1 L. Enzyme sample is diluted in buffer and preheated to reaction temperature. Substrate solution, 1% esterified pectin in water, is preheated to reaction temperature; reaction is started by adding 100 μL of diluted enzyme to 900 μL of substrate solution. Standards contain 100 μL methanol (0 to 100 mM in water) and 900 μL of substrate solution. After appropriate incubation time, samples are mixed and aliquot is injected into a gas chromatograph; peak areas of samples are compared to that of standards. One unit is amount of enzyme that produces one micromole of methanol per minute. (Adapted from Bartolome et al. (1972), J. Agric. Food Chem. 20 (2), 262-266.)


16.16 Assay Procedure CU10: Colorimetric Assay of Cellobiose Dehydrogenase, Measuring Reduction of DCIP

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 μL of diluted enzyme sample is added to 10 μL of 48 mM sodium fluoride (made by dissolving 2 mg NaF in 10 mL water), 10 μL of 3.6 mM 2,6-dichloroindophenol (DCIP, made by dissolving 9.6 mg in 10 mL water) and 80 μL 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 clear microtiter flat-bottomed plate and preheated to the appropriate temperature in a dry bath heater. Reaction is started by addition of 120 μL of 360 mM lactose (made by dissolving 1.23 g lactose in 100 mL water). Blank contains 10 μL sample, 10 μL 48 mM NaF, 10 μL 3.6 mM DCIP, 80 μL reaction buffer and 120 μL water. Absorbance at 520 nm is continuously monitored and compared to the molar absorptivity coefficient of DCIP. One unit is the amount of enzyme that reduces one micromole of DCIP per minute under the specified assay conditions. (Adapted from Baminger et al. (2001), Appl Environ Microbiol, 67(4), 1766-1774.)


16.17 Assay Procedure CU11: Colorimetric Assay of Aldolactonase, Measuring Glucono-Delta-Lactone

Enzyme sample is diluted in 50 mM acetate reaction buffer, pH 5 (made by dissolving 2.88 mL 99.7% glacial acetic acid in water, adjusting pH with 10 M NaOH and diluting to 1 L) and preheated to 37° C. 50 mL of 4.0 M hydroxylamine hydrochloride (made by dissolving 27.6 g hydroxylamine hydrochloride in water and diluting to 100 mL) is mixed with 50 mL of 3.0 M sodium hydroxide (made by dissolving 12 g of sodium hydroxide and diluting to 100 mL); the resulting alkaline hydroxylamine solution is used within the next 3 hours. 0.239 g of glucono-delta-lactone are dissolved in 100 mL reaction buffer that has been preheated to 37° C., and 125 μL of the resulting 13.4 mM substrate solution is immediately pipetted to a clear flat-bottomed microtiter plate. The reaction is started by addition of 15 μL diluted sample to substrate solution. Standards contain 80-125 μL of substrate solution, with the volume made up to 140 μL with reaction buffer. After 10 minutes incubation, 28 μL alkaline hydroxylamine solution is added, then 14 μL 4 M HCl is added (made by diluting concentrated HCl threefold in water), then 14 μL of 0.5 M FeCl3 (made by dissolving 8.1 g FeCl3 in water and diluting to 100 mL) is added. Absorbances are read at 540 nm and compared to the standard curve. One unit is the amount of enzyme that removes one micromole of glucono-delta-lactone per minute. (Adapted from Hestrin et al. (1949), J. Biol. Chem. 180, 249-261.)


16.18 Activity-Temperature Profiles

Temperature optima are determined by first determining the range of enzyme concentration that reproducibly displays initial velocity kinetics at 40° C. 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.


Example 17
Identification of Genes that Encode Secreted Proteins

Genes (and polypeptides) from the organisms Scytalidium thermophilum (Scyth), Myriococcum thermophilum (Myrth), and Aureobasidium pullulans (Aurpu) 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 18
Improvement of Thermophilic Cellulase Mixture by Various Proteins in an MTP Activity Assay Using aCS as Substrate

(Hemi-)cellulosic proteins of interest were cloned and expressed in A. niger as described above in Examples 8-10. Supernatants of protein MTP fermentations were added to a TEC-210 cellulase enzyme base mix as described above (Example 13), and acid pretreated corn stover (aCS) was used as the substrate. Several proteins demonstrated increased sugar release, as seem below in Table 7.









TABLE 7







Effect of various proteins spiked on TEC-210 using aCS substrate


in MTP assay











Target ID
SEQ ID NOs:
Glucose (AU)







TEC only

32.6



Scyth2p4_010825
231, 516, 801
36.8



Scyth2p4_008294
153, 438, 723
37.0



Scyth2p4_006005
110, 395, 680
37.0



MYRTH_3_00099
1054, 1360, 1666
37.0



Myrth2p4_006397
1029, 1335, 1641
37.1



MYRTH_2_03760
897, 1203, 1509
43.3



AURPU_3_00017
1775, 2162, 2549
36.8



AURPU_3_00429
1814, 2201, 2588
37.0



AURPU_3_00353
1848, 2235, 2622
37.3










In a second set of experiments with acid pretreated corn stover (aCS) as the substrate, supernatants of a different set of protein fermentations were added to TEC-210 as described above. Several proteins demonstrated increased sugar release, as shown below in Table 8.









TABLE 8







Effect a different set of various proteins spiked on TEC-210 using


aCS substrate











Target ID
SEQ ID NO:
Glucose (AU)







TEC only

52.6



Scyth2p4_009823
201, 486, 771
56.5



SCYTH_1_02579
258, 543, 828
57.1



SCYTH_1_00740
12, 297, 582
57.7



Myrth2p4_008437
1088, 1394, 1700
57.7



Myrth2p4_003274
931, 1237, 1543
58.3



Myrth2p4_006213
1107, 1413, 1719
70.5



AURPU_3_00208
2118, 2505, 2892
60.7



Aurpu2p4_008503
1970, 2357, 2744
61.5



Aurpu2p4_006782
1920, 2307, 2694
62.3










In a third set of experiments with aCS as the substrate, supernatant of GH61 MTP fermentations was added to a 3 enzyme cellulase base mixes, as described above. Spiking showed increased sugar release, as shown below in Table 9.









TABLE 9







Effect of various GH61 enzymes spiked


on 3 enzyme mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (AU)















3 enzyme mix

21.9



SCYTH_1_00672
104, 389, 674
26.6



SCYTH_1_05851
157, 442, 727
26.8



Scyth2p4_002689
50, 335, 620
27.6



MYRTH_2_03236
857, 1163, 1469
26.8



MYRTH_2_01413
953, 1259, 1565
27.1



MYRTH_2_03391
950, 1256, 1562
27.1



AURPU_3_00402
2001, 2388, 2775
24.0



AURPU_3_00407
2010, 2397, 2784
24.7



AURPU_3_00395
1904, 2291, 2678
26.3










In another set of experiments with acid pretreated corn stover (aCS) as the substrate, the supernatants of one protein MTP fermentations was added to TEC-210 as described above. This protein showed increased sugar release, as shown below in Table 10.









TABLE 10







Effect of the AURPU_3_00184 protein


spiked on TEC-210 using aCS substrate











Target ID
SEQ ID NOs:
Glucose (AU)















TEC only

25.1



AURPU_3_00184
1902, 2289, 2676
28.9










Example 19
Improvement of Thermophilic Cellulase Mixture by Various Scytalidium thermophilum Proteins in an Activity Assay at Labscale Including Mixing


Scytalidium thermophilum proteins were cloned and expressed in A. niger as described above (Examples 8-10). Concentrated supernatants from shake flask fermentations were used in sugar release activity assays as described above (Example 14), using 10% aCS NREL as feedstock. In one set of experiments, supernatant of the Scytalidium thermophilum protein Scyth2p4009442 was spiked based on protein dosage on top of a TEC-210 base mix, as described above. The protein showed increased sugar release, as shown below in Table 11.









TABLE 11







Effect of a Scytalidium thermophilum protein spiked based


on protein dosage on TEC-210 using 10% DM aCS substrate










Glucose











Target ID
SEQ ID NOs:
Average (AU)
stdev













TEC only

31.4
0.07


Scyth2p4_009442
178, 463, 748
32.5
0.09









Example 20
Improvement of Thermophilic Cellulase Mixture by Various Aureobasidium pullulans Proteins in an Activity Assay at Labscale Including Mixing

The cellulase enhancing activity of various Aureobasidium pullulans beta-galactosidase (BG) proteins were further analyzed. The supernatant of the A. niger expressing shake flask fermentations were concentrated and spiked in a dosage of 0.45 mg/gDM on top of a base activity of a three enzyme base mix (4.55 mg/gDM composed of: CBHI at 1.25 g/gDM, CBHII at 1.5 mg/gDM and GH61 at 1.8 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15). Addition of the Aureobasidium pullulans BG proteins yielded increased sugar release, as shown below in Table 12.









TABLE 12







Effect of Aureobasidium pullulans BG proteins


spiked on top of a 3E mix using aCS substrate











Protein ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

6.7



Aurpu2p4_006782
1920, 2307, 2694
26.1



AURPU_3_00208
2118, 2505, 2892
26.8










Example 21
Improvement of Thermophilic Cellulase Mixture by Various Proteins in an Activity Assay at Labscale Including Mixing

The cellulase enhancing activity of various GH61 proteins were further analyzed. The supernatant of the A. niger expressing Scyth2p4002220, MYRTH204272, and MYRTH201413 shake flask fermentations were concentrated and spiked in a dosage of 1.8 mg/gDM on top of a base activity of a three enzyme base mix (3.2 mg/gDM composed of: BG at 0.45 g/gDM, CBHI at 1.5 mg/gDM and CBHII at 1.25 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15). Addition of these GH61 proteins yielded increased sugar release, as shown below in Table 13.









TABLE 13







Effect of various GH61 proteins spiked


on top of a 3E mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

29.7



Scyth2p4_002220
42, 327, 612
32.5



MYRTH_2_04272
1040, 1346, 1652
32.1



MYRTH_2_01413
953, 1259, 1565
32.4










In another experiment, the cellulase enhancing activity of Scytalidium thermophilum CBHII protein SCYTH103721 was further analyzed. The SCYTH103721 gene was cloned and expressed in A. niger as described above (Examples 8-10). The supernatant of an A. niger expressing SCYTH103721 shake flask fermentation was concentrated and spiked in a dosage of 1.5 mg/gDM on top of a base activity of a three enzyme base mix (3.5 mg/gDM composed of: BG at 0.45 g/gDM, CBHI at 1.25 mg/gDM and GH61 at 1.8 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15). Addition of the SCYTH103721 protein yielded increased sugar release, as shown below in Table 14.









TABLE 14







Effect of CBHII SCYTH_1_03721 protein spiked


on top of a 3E mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

28.1



SCYTH_1_03721
129, 414, 699
32.5










In another experiment, the cellulase enhancing activity of another Myriococcum thermophilum GH61 protein was further analysed. The supernatant of the A. niger expressing MYRTH203760 shake flask fermentation was concentrated and spiked in a dosage of 1.8 mg/gDM on top of a base activity of a three enzyme base mix (3.2 mg/gDM composed of: BG at 0.45 g/gDM, CBHI at 1.5 mg/gDM and CBHII at 1.25 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15). Addition of this Myriococcum thermophilum GH61 protein yielded increased sugar release, as shown below in Table 15.









TABLE 15







Effect of GH61 protein MYRTH_2_03760 spiked


on top of a 3E mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

17.8



MYRTH_2_03760
897, 1203, 1509
19.1










In another experiment, the cellulose-enhancing activity of Myriococcum thermophilum CBHI protein MYRTH2p4003203 was further analyzed. The MYRTH2p4003203 gene was cloned and expressed in A. niger as described above (Examples 8-10). The supernatant of an A. niger expressing MYRTH2p4003203 shake flask fermentation was concentrated and spiked in a dosage of 1.25 mg/gDM on top of a base activity of a three enzyme base mix (3.75 mg/gDM composed of: BG at 0.45 g/gDM, CBHII at 1.5 mg/gDM and GH61 at 1.8 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15).


Addition of this Myriococcum thermophilum CBHI protein yielded increased sugar release, as shown below in Table 16.









TABLE 16







Effect of CBHI MYRTH2p4_003203 protein


spiked on top of a 3E mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

19.2



MYRTH2p4_003203
930, 1237, 1543
22.1










In another experiment, the cellulase enhancing activity of Myriococcum thermophilum beta-galactosidase (BG) protein MYRTH100021 was further analyzed. The supernatant of an A. niger expressing MYRTH100021 shake flask fermentation was concentrated and spiked in a dosage of 0.45 mg/gDM on top of a base activity of a three enzyme base mix (4.55 mg/gDM composed of: CBHI at 1.25 g/gDM, CBHII at 1.5 mg/gDM and GH61 at 1.8 mg/gDM) at a feedstock concentration of 10% (w/w) aCS, as described above (Example 14). As a negative control, the 3 enzyme base mix was also tested. All experiments were performed at least in duplicate and were incubated for 72 hours at 65° C. in an oven incubator (Techne HB-1D hybridization oven) while rotating at set-point 3. After incubation, the samples were centrifuged and soluble sugars were analysed by HPLC as described above (Example 15).


Addition of this Myriococcum thermophilum BG protein yielded increased sugar release, as shown below in Table 17 and in FIG. 8.









TABLE 17







Effect of beta-galactosidase MYRTH_1_00021 protein


spiked on top of a 3E mix using aCS substrate











Target ID
SEQ ID NOs:
Glucose (g/L)















3 enzyme mix

13.8



MYRTH_1_00021
1113, 1419, 1725
16.6










Example 22
Identification of Thermophilic Various Arabino(Furano)Sidases

The arabino(furano)sidase activity of various enzymes was further analysed, as described above (Example 16.1). The supernatant of A. niger shake flask fermentations were concentrated and assayed for arabinose release from wheat arabinoxylan, which was pre-digested by an endo-xylanase, after incubation for 24 hours at pH 4.5 and 65° C. Three enzymes showed increased arabinose release as shown below in Table 18.









TABLE 18







Effect of various proteins on pre-


digested wheat arabinoxylan substrate












μg/mL
% arabinose


Target ID
SEQ ID NOs:
arabinose
release of max













no enzyme

4.5
0


SCYTH_1_01777
36, 321, 606
7.6
0.4


SCYTH_1_01831
191, 476, 761
23.4
2.7


MYRTH_1_00007
1015, 1321, 1627
10.6
0.9


MYRTH_3_00127
1144, 1450, 1756
161.4
22


MYRTH_1_00002
1109, 1415, 1721
5.3
0.1


MYRTH_2_00959
1126, 1432, 1738
149.0
20.6


AURPU_3_00341
1976, 2363, 2750
7.8
0.5


AURPU_3_00342
1824, 2211, 2598
7.3
0.4


AURPU_3_00410
1992, 2379, 2766
11.7
1


AURPU_3_00333
1993, 2380, 2767
5.6
0.2









Example 23
Identification of Thermophilic Beta-Xylosidases

The beta-xylosidase activity of various enzymes was further analyzed. The supernatants of the A. niger shake flask fermentations were concentrated and assayed in different dosages for xylose release from xylobiose after incubation for 24 hours at pH 4.5 and 65° C. as described above (Example 16.2). Several enzymes showed significant xylose release from xylobiose as shown below in Table 19.









TABLE 19







Effect of various enzymes on release of xylose from xylobiose












Concen-
% xylose release




tration
from xylobiose (%


Target ID
SEQ ID NOs:
(w/w)
from max possible)













Scyth2p4_001371
19, 304, 589
0.1%
6





1%

22


MYRTH_1_00003
1138, 1444, 1750
0.1%
0





1%

1


MYRTH_2_01280
1131, 1437, 1743
0.1%
0





1%

4


MYRTH2p4_001496
894, 1200, 1506
0.5%
9


MYRTH_2_00959
1126, 1433, 1739
0.5%
1


AURPU_3_00184
1902, 2289, 2676
0.1%
57





1%

100









Example 24
Identification of Thermophilic Scytalidium thermophilum Acetyl-Xylan Esterase

The acetyl-xylan esterase activity of Scytalidium thermophilum SCYTH207393 was further analyzed. The supernatant of this Scytalidium thermophilum A. niger shake flask fermentation was concentrated and assayed for acetic acid release from acid pretreated corn stover as described above (Example 16.3). The enzymes was identified as active acetyl xylan esterase because it was able to release acetic acid from the substrate as is shown in Table 20.









TABLE 20







Effect of SCYTH_2_07393 (SEQ ID NOs: 262, 547, 832) enzyme


on release of acetic acid from pretreated corn stover










SCYTH_2_07393
Acetic acid (μg/mL)














no enzyme
63



0.1% (w/w)
131



1% (w/w)
152










Example 25
Characterization Various Thermophilic Endoxylanases

The endoxylanase activity of SCYTH109019, SCYTH109441, SCYTH101114, MYRTH203560, AURPU300013, and AURPU300019 proteins was further analyzed. The supernatant of the A. niger shake flask fermentations were concentrated and assayed for endoxylanase activity on wheat arabinoxylan oligosaccharides and beech wood xylan as described above in endoxylanase activity assay 1 (Example 16.4). The proteins were able to release xylose and xylose oligomers release from the two substrates after incubation for 24 hours with 1% (w/w) enzyme dose at pH 4.5 and 65° C. as is shown in Table 21.









TABLE 21







Effect of various proteins on release of xylose and xylose oligomers


from Beech wood xylan and Wheat arabinoxylan









Amount released (μg/mg substrate)














1% (w/w) E/S
SEQ ID NOs:
xylose
xylobiose
xylotriose
xylotetraose

















Beech wood
no enzyme

1.4
0.3
0.0
0.2


xylan
SCYTH_1_09019
260, 545, 830
63.0
373.0
10.7
0.5



SCYTH_1_09441
155, 440, 725
24.4
129.9
140.6
30.2



SCYTH_1_01114
218, 503, 788
29.3
180.4
162.5
20.5



MYRTH_2_03560
1022, 1328, 1634
7.0
383.9
7.8
0.8



AURPU_3_00013
1924, 2311, 2698
84.6
337.1
53.4
0.5


ara
no enzyme

0.5
0.0
0.0
0.1


bin
SCYTH_1_09019
260, 545, 830
43.6
75.3
1.9
0.0



SCYTH_1_09441
155, 440, 725
5.7
14.3
7.3
2.0



SCYTH_1_01114
218, 503, 788
8.0
18.4
8.3
1.4



MYRTH_2_03560
1022, 1328, 1634
4.1
73.4
2.3
0.0



AURPU_3_00013
1924, 2311, 2698
55.8
87.0
1.0
0.0



AURPU_3_00019
1925, 2312, 2699
1.7
4.3
5.7
2.6









In a second set of experiments, the endoxylanase activity of the proteins SCYTH109019, SCYTH100286, SCYTH109441, SCYTH101114, MYRTH203560, MYRTH201976, AURPU300013, AURPU300019, AURPU300018 was further analyzed as described above in endoxylanase activity assay 2 (Example 16.5). The supernatant of the A. niger shake flask fermentations were concentrated and assayed for endoxylanase activity by measuring reducing-end formation expressed as xylose equivalents after incubation of the enzymes at 0.1% (w/w) dose on wheat arabinoxylan during 24 hours at 65° C. and pH 4.5. The enzymes were able to release reducing sugars from the substrates, as shown in Table 22 and in FIG. 2, where panels A, B and C correspond to proteins from Scytalidium thermophilum, Myriococcum thermophilum, and Aureobasidium pullulans, respectively.









TABLE 22







Effect of various proteins on the release of reducing sugars


(reported as xylose equivalents) from Wheat arabinoxylan









reducing-ends expressed in xylose equivalents (μg/mL)
















Target ID
SEQ ID NOs:
t = 0 h
t = 0.5 h
t = 1 h
t = 2 h
t = 3 h
t = 4 h
t = 6 h
t = 24 h



















no enzyme

−6.0
−14.5
−17.0
−11.9
−11.7
−12.6
−11.8
−9.9


SCYTH_1_09019
260, 545, 830
2.3
340.0
394.0
438.0
440.1
440.2
446.4
462.1


SCYTH_1_00286
237, 522, 807
2.3
0.8
1.9
0.4
3.6
5.0
9.1
26.7


SCYTH_1_09441
155, 440, 725
−6.0
187.1
213.4
240.1
245.8
244.7
241.4
248.4


SCYTH_1_01114
218, 503, 788
−6.0
251.3
248.2
243.7
237.6
244.8
257.0
266.4


MYRTH_2_03560
1022, 1328, 1634
2.3
292.5
344.9
391.8
415.2
437.7
457.9
454.6


MYRTH_2_01976
972, 1278, 1584
−6.0
236.9
242.3
224.9
235.1
214.4
220.3
211.2


AURPU_3_00013
1924, 2311, 2698
2.3
149.8
267.0
380.5
411.1
439.5
475.6
548.5


AURPU_3_00019
1925, 2312, 2699
−6.0
62.0
86.3
110.2
122.1
122.8
131.9
127.4


AURPU_3_00018
2108, 2495, 2882
−6.0
165.9
169.9
190.1
206.6
222.1
249.5
240.6









Example 26
Characterization of Thermophilic Aureobasidium pullulans Xyloglucanase

The xyloglucanase activity of AURPU300030 (SEQ ID NOs: 1778, 2165, 2552) and AURPU300028 (SEQ ID NOs: 1947, 2334, 2721) proteins were further analyzed. The supernatant of these two Aureobasidium pullulan A. niger shake flask fermentations were concentrated and assayed for xyloglucanase activity on Tamarind xyloglucan as described above (Example 16.6). Both enzymes were identified as active xyloglucanase because they were able to release low molecular weight oligosaccharides, as shown in FIG. 3.


Example 27
Further Characterization of Expressed Enzymes from Scytalidium thermophilum

The Scytalidium thermophilum proteins SCYTH207268, SCYTH207393, SCYTH100740, SCYTH103721, SCYTH103688, SCYTH101623, Scyth2p4005037, and SCYTH207965 were further characterized using the assay protocols and assay conditions indicated in the table below.



















SEQ ID


Assay
Activity
Fold increase


Target ID
NOs:
Assay Protocol
Substrate
Conditions
(U/mL)
over control*





















SCYTH_2_07268
174, 459,
CU5
Ethyl ferulate, 4 mM
pH 5.3,
7
na



744
(Example 16.11)

40° C.,






30 min


SCYTH_2_07393
262, 547,
CU8
acetylated xylan from
pH 5,
3.1
na



832
(Example 16.14)
beechwood 0.4%
40° C.,






15 min


SCYTH_1_00740
12, 297,
CU1
4-nitrophenyl beta-D-
pH 5,
3.3
na



582
(Example 16.7)
glucosaminide, 1 mM
40° C.,






30 min


SCYTH_1_03721
129, 414,
CU7
4-methylumbelliferyl
pH 5,
0.002
na



699
(Example 16.13)
beta-D-lactoside, 0.2 mM
40° C.,






30 min


SCYTH_1_03688
259, 544,
CU1
4-nitrophenyl alpha-
pH 5,
1.3
65



829
(Example 16.7)
L-arabinofuranoside,
40° C.,





1 mM
30 min


SCYTH_1_01623
160, 445,
CU7
4-methylumbelliferyl
pH 5,
0.0066
6.6



730
(Example 16.13)
beta-D-cellobioside,
40° C.,





0.2 mM
30 min


Scyth2p4_005037
86, 371,
CU6
Polygalacturonic
pH 8,
0.43
na



656
(Example 16.12)
acid, 0.9%
40° C.,






initial rate


SCYTH_2_07965
125, 410,
CU6
Rhamnogalacturonan
pH 6,
1.1
na



695
(Example 16.12)
I, 0.7%
40° C.,






initial rate





*na, not applicable as control exhibited no detectable activity. Control is an equal volume of supernatant from a vector-only transformant



U, micromole product formed per minute under the indicated assay conditions







Example 28
Further Characterization of Expressed Enzymes from Myriococcum thermophilum

The Myriococcum thermophilum proteins Myrth2p4003495, Myrth2p4005155, Myrth2p4007061, MYRTH201934, MYRTH2p4001537, MYRTH2p4005923, MYRTH2p4003942, MYRTH100080, MYRTH409372, MYRTH2p4001451, MYRTH409820, Myrth2p4003941, MYRTH100024, MYRTH2p4002293, MYRTH300003, MYRTH300097, MYRTH406111, Myrth2p4001304, Myrth2p4000359, Myrth2p4007801, MYRTH2p4003203, and Myrth2p4006226 were further characterized using the assay protocols and assay conditions indicated in the table below.
























Fold








increase



SEQ ID


Assay
Activity
over


Target ID
NOs:
Assay Protocol
Substrate
Conditions
(U/mL)
control*





















Myrth2p4_003495
934, 1240,
CU11
glucono-delta-lactone
pH 5,
55
12.2



1546
(Example 16.17)
12 mM
37° C.,






30 min


Myrth2p4_005155
982, 1288,
CU4
alpha-D-Glucose,
pH 5,
83
na



1594
(Example 16.12)
10 umol/mL
40° C.,






continuous


Myrth2p4_007061
1045, 1351,
CU4
alpha-D-Glucose,
pH 5,
96
na



1657
(Example 16.10)
10 umol/mL
40° C.,






continuous


MYRTH_2_01934
980, 1286,
CU3
alpha-naphthyl acetate,
pH 5,
11.8
na



1592
(Example 16.9)
0.4 mM
30° C.,






continuous


MYRTH2p4_001537
895, 1201,
CU8
acetylated xylan from
pH 5,
2.7
na



1507
(Example 16.14)
beechwood 0.4%
40° C.,






15 min


MYRTH2p4_005923
1013, 1319,
CU8
acetylated xylan from
pH 5,
3.5
na



1625
(Example 16.14)
beechwood 0.4%
40° C.,






15 min


MYRTH2p4_003942
944, 1250,
CU9
esterified pectin, 1%
pH 8,
34
na



1556
(Example 16.15)

40° C.,






15 min


MYRTH_1_00080
1119, 1425,
CU1
4-nitrophenyl acetate, 1 mM
pH 5,
0.2
5



1731
(Example 16.7)

30° C.,






30 min


MYRTH_4_09372
1146, 1452,
CU2
Xylan from beechwood
pH 5,
17.3
58



1758
(Example 16.8)
0.2%
40° C.,






30 min


MYRTH2p4_001451
889, 1195,
CU2
Xylan from beechwood
pH 5,
12.7
42



1501
(Example 16.8)
0.2%
40° C.,






30 min


MYRTH_4_09820
1147, 1453
CU2
Carboxymethylcellulose,
pH 5,
3
10



1759
(Example 16.8)
0.2%
40° C.,






30 min


Myrth2p4_003941
943, 1249,
CU2
Laminarin, 0.2%
pH 5,
2.2
220



1555
(Example 16.8)

40° C.,






30 min


MYRTH_1_00024
943, 1249,
CU2
Lichenan, 0.2%
pH 5,
1.05
11.7



1555
(Example 16.8)

40° C.,






30 min


MYRTH2p4_002293
908, 1214,
CU2
Carboxymenthylcellulose,
pH 5,
4.1
13.7



1520
(Example 16.8)
0.2%
40° C.,






30 min


MYRTH_3_00003
1138, 1444,
CU2
Locust bean gum, 0.2%
pH 5,
1.6
1600



1750
(Example 16.8)

40° C.,






30 min


MYRTH_3_00097
974, 1280,
CU2
Carboxymethylcellulose,
pH 5,
2
6.7



1586
(Example 16.8)
0.2%
40° C.,






30 min


MYRTH_4_06111
1071, 1377,
CU2
Carboxymethylcellulose,
pH 5,
16
53



1683
(Example 16.8)
0.2%
40° C.,






30 min


Myrth2p4_001304
876, 1182,
CU10
Lactose, 180 mM
pH 5,
0.37
na



1488
(Example 16.16)

40° C.,






continuous


Myrth2p4_000359
858, 1164,
CU10
Lactose, 180 mM
pH 5,
0.43
na



1470
(Example 16.16)

40° C.,






continuous


Myrth2p4_007801
1065, 1371,
CU6
Polygalacturonic acid,
pH 8,
1.1
na



1677
(Example 16.12)
0.9%
40° C.,






continuous


MYRTH2p4_003203
930, 1236,
CU7
4-methylumbelliferyl
pH 5,
0.03
30



1542
(Example 16.13)
beta-D-cellobioside
40° C.,






30 min


Myrth2p4_006226
1026, 1332,
CU6
Polygalacturonic acid
pH 8,
10
na



1638
(Example 16.12)
0.9%
40° C.,






continuous





*na, not applicable as control exhibited no detectable activity. Control is an equal volume of supernatant from a vector-only transformant



U, micromole product formed per minute under the indicated assay conditions







Example 29
Further Characterization of Expressed Enzymes from Aureobasidium pullulans

The Aureobasidium pullulans proteins Aurpu2p4002220, Aurpu2p4008140, Aurpu2p4010203, Aurpu2p4009597, Aurpu2p4009401, AURPU300030, AURPU300153, AURPU300155, AURPU300166, AURPU300175, AURPU300177, AURPU300191, AURPU300241, AURPU300284, AURPU300296, AURPU300035, and Aurpu2p4011071, were further characterized using the assay protocols and assay conditions indicated in the table below.
























Fold








increase



SEQ ID
Assay

Assay
Activity
over


Target ID
NOs:
Protocol
Substrate
Conditions
(U/ml)
control*





















Aurpu2p4_002220
1836, 2223,
CU4
alpha-D-Glucose, 10 umol/mL
pH 5, 40° C.,
39
na



2610
(Example 16.10)

continuous


Aurpu2p4_008140
1959, 2346,
CU5
Ethyl ferulate, 4 mM
pH 5.3,
121
na



2733
(Example 16.11)

40° C., 30 min


Aurpu2p4_010203
2014, 2401,
CU1
4-nitrophenyl acetate, 1 mM
pH 5, 30° C.,
0.17
4.3



2788
(Example 16.7)

30 min


Aurpu2p4_009597
1995, 2382,
CU3
alpha-naphthyl acetate, 0.4 mM
pH 5, 30° C.,
48
na



2769
(Example 16.9)

continuous


Aurpu2p4_009401
1989, 2376,
CU1
4-nitrophenyl butyrate, 1 mM
pH 7, 30° C.,
1.2
na



2763
(Example 16.7)

30 min


AURPU_3_00030
1778, 2165,
CU2
xyloglucan from tamarind,
pH 5, 40° C.,
7.6
109



2552
(Example 16.8)
0.08%
30 min


AURPU_3_00153
1987, 2374,
CU1
4-nitrophenyl alpha-L-
pH 5, 40° C.,
0.51
na



2761
(Example 16.7)
arabinopyranoside, 1 mM
30 min


AURPU_3_00155
1952, 2339,
CU2
polygalacturonic acid, 0.1%
pH 5, 40° C.,
157
390



2726
(Example 16.8)

30 min


AURPU_3_00166
1893, 2280,
CU2
polygalacturonic acid, 0.1%
pH 5, 40° C.,
4.1
10.3



2667
(Example 16.8)

30 min


AURPU_3_00175
1978, 2365,
CU2
polygalacturonic acid, 0.1%
pH 5, 40° C.,
12
30



2752
(Example 16.8)

30 min


AURPU_3_00177
1934, 2321,
CU2
polygalacturonic acid, 0.1%
pH 5, 40° C.,
2.6
6.5



2708
(Example 16.8)

30 min


AURPU_3_00191
1873, 2260,
CU1
4-nitrophenyl beta-D-
pH 5, 40° C.,
20.6
412



2647
(Example 16.7)
glucopyranoside, 1 mM
30 min


AURPU_3_00241
1936, 2323,
CU1
4-nitrophenyl beta-D-
pH 5, 40° C.,
89.8
1800



2710
(Example 16.7)
glucopyranoside, 1 mM
30 min


AURPU_3_00284
1801, 2188,
CU2
sucrose, 0.2%
pH 5, 40° C.,
10.4
210



2575
(Example 16.8)

30 min


AURPU_3_00296
1847, 2234,
CU2
sucrose, 0.2%
pH 5, 40° C.,
12.6
250



2621
(Example 16.8)

30 min


AURPU_3_00035
1931, 2318,
CU1
4-nitrophenyl alpha-L-
pH 5, 40° C.,
53
na



2705
(Example 16.7)
arabinopyranoside, 1 mM
30 min


Aurpu2p4_011071
2040, 2427,
CU6
Rhamnogalacturonan I, 0.7%
pH 6, 40° C.,
0.86
na



2814
(Example 16.12)

continuous





*na, not applicable as control exhibited no detectable activity. Control is an equal volume of supernatant from a vector-only transformant



U, micromole product formed per minute under the indicated assay conditions







Example 30
Determination of Activity-Temperature Profiles

The activity-temperature profiles were determined for various proteins of the present invention according to the protocol in Example 16.18. Results for are shown in FIGS. 4-16 for various proteins from Scytalidium thermophilum, Myriococcum thermophilum, and Aureobasidium pullulans, using the Assay Protocols and Assay Conditions indicated below in Tables 23-25.









TABLE 23







Activity-temperature profiles for various Scytalidium thermophilum proteins













SEQ ID
Assay



Figure
Protein
NOs:
protocol
Assay conditions













4A
SCYTH_1_09019
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 6.0, 30 min


4B
SCYTH_1_01114
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 5.5, 30 min


4C
SCYTH_1_09441
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 5.5, 30 min


5A
Scyth2p4_009303
CU1
1 mM pNP-alpha-L-Arabinofuranoside,




(Example 16.7)
pH 5.0, 30 min


5B
Scyth2p4_004025
CU2
0.1% wheat arabinoxylan,




(Example 16.8)
low viscosity, pH 5.0, 30 min.


5C
SCYTH_1_00574
CU2
0.2% carboxymethylcellulose,




(Example 16.8)
pH 6.0, 30 min


5D
SCYTH_1_08979
CU7
0.2 mM 4-methylumbelliferyl-




(Example 16.13)
cellobioside, pH 5.0, 30 min
















TABLE 24







Activity-temperature profiles for various Myriococcum thermophilum proteins













SEQ ID




Figure
Protein
NOs:
Assay protocol
Assay conditions













6A
MYRTH_2_03560
CU2
0.2% beechwood xylan,




(Example 16.8)
pH 4.0, 30 min


6B
MYRTH_2_04091
CU2
0.2% beechwood xylan




(Example 16.8)
pH 7.0, 30 min


6C
MYRTH_1_00068
CU2
0.2% beechwood xylan




(Example 16.8)
pH 4.0, 30 min


6D
MYRTH_2_00256
CU7
4-methylumbelliferyl-




(Example 16.13)
cellobioside, pH 5.5, 30 min


7A
MYRTH_2_01976
CU2
0.2% beechwood xylan




(Example 16.8)
pH 6.0, 30 min


7B
MYRTH_2_00218
CU2
0.2% carboxymethylcellulose




(Example 16.8)
(7M + 4M), pH 5, 30 min


7C
MYRTH_1_00018
CU1
1 mM 4-nitrophenyl beta-D-




(Example 16.7)
galactopyranoside, pH 4.0, 30 min


7D
MYRTH_2_04288
CU2
0.08% locust bean gum,




(Example 16.8)
pH 5.0, 30 min


8A
MYRTH_2_04289
CU2
0.08% locust bean gum,




(Example 16.8)
pH 5.0, 30 min


8B
MYRTH2p4_001339
CU1
1 mM pNP-beta-glucopyranoside,




(Example 16.7)
pH 5.0, 30 min


8C
MYRTH_1_00021
CU1
1 mM pNP beta-glucopyranoside,




(Example 16.7)
pH 5.5, 30 min


8D
MYRTH_2_00959
CU2
0.1% low viscosity wheat




(Example 16.8)
arabinoxylan, pH 6.0, 30 min


9A
MYRTH_1_00035
CU2
0.08% locust bean gum,




(Example 16.8)
pH 6.0, 30 min


9B
MYRTH2p4_005976
CU2
0.2% carboxymethylcellulose,




(Example 16.8)
pH 5.5, 30 min


9C
MYRTH_4_03993
CU7
4-methylumbelliferyl-cellobioside,




(Example 16.13)
pH 4.5, 30 min


9D
MYRTH_3_00099
CU7
4-methylumbelliferyl-lactoside,




(Example 16.13)
pH 4.0, 30 min


10A 
MYRTH_2_00848
CU1
1 mM p-nitrophenyl-alpha-L-




(Example 16.7)
arabinopyranoside, pH 5.0, 30 min


10B 
MYRTH_3_00127
CU2
0.1% wheat arabinoxylan, low




(Example 16.8)
viscosity, pH 4.5, 30 min


10C 
Myrth2p4_006408
CU2
0.08% xyloglucan,




(Example 16.8)
pH 6.0, 30 min


10D 
MYRTH2p4_001496
CU1
1 mM p-nitrophenyl-beta-D-




(Example 16.7)
Xylopyranoside, pH 4.5, 30 min


11A 
MYRTH_2_00256
CU7
0.2 mM 4-methylumbelliferyl-




(Example 16.13)
cellobioside, pH 5.0, 30 min
















TABLE 25







Activity-temperature profiles for various Aureobasidium pullulans proteins













SEQ ID




Figure
Protein
NOs:
Assay protocol
Assay conditions













12A
AURPU_00052
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 4.5, 30 min


12B
AURPU_3_00014
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 4.0, 30 min


12C
Aurpu2p4_005858
CU1
5 mM pNP-beta-D Glucopyranoside,




(Example 16.7)
pH 5.0, 30 min


12D
Aurpu2p4_010898
CU1
5 mM pNP-N-acetyl-beta-D-




(Example 16.7)
glucosaminide, pH 4.0, 30 min


13A
AURPU_3_00307
CU1
(1 mM pNP-Beta-Galactopyranoside,




(Example 16.7)
pH 4.0, 30 min) GH20


13B
Aurpu2p4_008021
CU2
(0.2% Beta-mannan,




(Example 16.8)
pH 5, 30 min) GH5


13C
Aurpu2p4_009751
CU2
(0.2% xylan from beechwood,




(Example 16.8)
pH 4.0, 30 min) GH10


13D
AURPU_3_00016
CU2
0.2% alpha-tomatine,




(Example 16.8)
pH 4.0, 30 min


14A
AURPU_3_00018
CU2
(0.2% xylan from beechwood




(Example 16.8)
pH 3.5, 30 min) GH 11


14B
AURPU_3_00019
CU2
(0.2% xylan from beechwood




(Example 16.8)
pH 3.5, 30 min) GH 11


14C
AURPU_3_00147
CU1
(1 mM 4-nitrophenyl beta-D-




(Example 16.7)
mannopyranoside, pH 3.0, 30 min) GH2


14D
Aurpu2p4_006782
CU1
(1 mM pNP-β-D Glucopyranoside,




(Example 16.7)
30 min, pH 4.0) GH 3


15A
AURPU_3_00192
CU1
(1 mM pNP-beta-Glucopyranoside,




(Example 16.7)
pH 4.0, 30 min) GH3


15B
AURPU_3_00208
CU1
(1 mM pNP-beta-Glucopyranoside,




(Example 16.7)
pH 4.0, 30 min) GH3


15C
Aurpu2p4_001633
CU2
(0.2% carboxymethylcellulose,




(Example 16.8)
pH 5.5, 30 min) GH5


15D
AURPU_ 3_00312
CU1
(1 mM pNP-beta-Glucopyranoside,




(Example 16.7)
pH 5.0, 30 min) GH5


16A
AURPU_3_00183
CU2
0.08% locust bean gum,




(Example 16.8)
pH 4.5, 30 min


16B
AURPU_3_00013
CU2
0.2% xylan from beechwood,




(Example 16.8)
pH 6.0, 30 min


16C
AURPU_3_00184
CU1
1 mM p-nitrophenyl-beta-d-




(Example 16.7)
xylopyranoside, pH 5.0, 30 min


16D
Aurpu2p4_011071
CU6
0.7% Rhamnogalacturonan I from




(Example 16.12)
potato, pH 5.0, initial rate









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.

Claims
  • 1. An isolated polypeptide which is: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 571-855, 1468-1773, or 2548-2934;(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: 286-570, 1162-1467, or 2161-2547;(d) a polypeptide comprising an amino acid sequence encoded by any one of 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 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).
  • 2. The isolated polypeptide of claim 1, wherein said polypeptide has a corresponding function and/or protein activity according to Tables 1A-1C.
  • 3. The isolated polypeptide of claim 1 or 2 comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 571-855, 1468-1773, or 2548-2934.
  • 4. The isolated polypeptide of any one of claims 1 to 3, wherein said polypeptide is a recombinant polypeptide.
  • 5. The isolated polypeptide of any one of claims 1 to 4 obtainable from a fungus.
  • 6. The isolated polypeptide of any one of claims 1 to 5, wherein said fungus is from the genus Scytalidium, Myriococcum, or Aureobasidium.
  • 7. The isolated polypeptide of any one of claims 1 to 6, wherein said fungus is Scytalidium thermophilum, Myriococcum thermophilum, or Aureobasidium pullulans.
  • 8. An antibody that specifically binds to the isolated polypeptide of any one of claims 1 to 7.
  • 9. An isolated polynucleotide molecule encoding the polypeptide of any one of claims 1 to 7.
  • 10. 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: 571-855, 1468-1773, or 2548-2934;(b) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-285, 856-1161, or 1774-2160;(c) a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 286-570, 1162-1467, or 2161-2547;(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).
  • 11. The isolated polynucleotide molecule of claim 9 or 10 obtainable from a fungus.
  • 12. The isolated polynucleotide molecule of claim 11, wherein said fungus is from the genus Scytalidium, Myriococcum, or Aureobasidium.
  • 13. The isolated polynucleotide molecule of claim 12, wherein said fungus is Scytalidium thermophilum, Myriococcum thermophilum, or Aureobasidium pullulans.
  • 14. A vector comprising a polynucleotide molecule as defined in any one of claims 9 to 13.
  • 15. The vector of claim 14 further comprising a regulatory sequence operatively linked to said polynucleotide molecule for expression of same in a suitable host cell.
  • 16. The vector of claim 15, wherein said suitable host cell is a bacterial cell.
  • 17. The vector of claim 15, wherein said suitable host cell is a fungal cell.
  • 18. The vector of claim 17, wherein said fungal cell is a filamentous fungal cell.
  • 19. A recombinant host cell comprising the polynucleotide molecule as defined in any one of claims 9 to 13, or a vector as defined in any one of claims 14 to 18.
  • 20. The recombinant host cell of claim 19, wherein said cell is a bacterial cell.
  • 21. The recombinant host cell of claim 19, wherein said cell is a fungal cell.
  • 22. The recombinant host cell of claim 21, wherein said fungal cell is a filamentous fungal cell.
  • 23. A polypeptide obtainable by expressing the polynucleotide molecule of any one of claims 9 to 13, or the vector of any one of claims 14 to 18 in a suitable host cell.
  • 24. A composition comprising the polypeptide of any one of claim 1 to 7 or 23, or the recombinant host cell of any one of claims 19 to 22.
  • 25. The composition of claim 24 further comprising a suitable carrier.
  • 26. The composition of claim 24 or 25 further comprising a substrate of said polypeptide.
  • 27. The composition of claim 26, wherein said substrate is biomass.
  • 28. A method for producing the polypeptide of any one of claim 1 to 7 or 23, said method comprising: (a) culturing a strain comprising the polynucleotide molecule of any one of claims 9 to 13 or the vector of any one of claims 14 to 18 under conditions conducive for the production of said polypeptide; and(b) recovering said polypeptide.
  • 29. The method of claim 28, wherein said strain is a bacterial strain.
  • 30. The method of claim 28, wherein said strain is a fungal strain.
  • 31. The method of claim 30, wherein said fungal strain is a filamentous fungal strain.
  • 32. A method for producing the polypeptide of any one of claim 1 to 7 or 23, said method comprising: (a) culturing the recombinant host cell of any one of claims 19 to 22 under conditions conducive for the production of said polypeptide; and(b) recovering said polypeptide.
  • 33. A method for preparing a food product, said method comprising incorporating the polypeptide of any one of claim 1 to 7 or 23 during preparation of said food product.
  • 34. The method of claim 33, wherein said food product is a bakery product.
  • 35. Use of the polypeptide of any one of claim 1 to 7 or 23 for the preparation or processing of a food product.
  • 36. The use of claim 33, wherein said food product is a bakery product.
  • 37. The polypeptide of any one of claim 1 to 7 or 23 for use in the preparation or processing of a food product.
  • 38. The polypeptide of claim 37, wherein said food product is a bakery product.
  • 39. Use of the polypeptide of any one of claim 1 to 7 or 23 for the preparation of animal feed.
  • 40. Use of the polypeptide of any one of claim 1 to 7 or 23 for increasing digestion or absorption of animal feed.
  • 41. The use of claim 39 or 40, wherein said animal feed is a cereal-based feed.
  • 42. The polypeptide of any one of claim 1 to 7 or 23 for the preparation of animal feed, or for increasing digestion or absorption of animal feed.
  • 43. The polypeptide of claim 42, wherein said animal feed is a cereal-based feed.
  • 44. Use of the polypeptide of any one of claim 1 to 7 or 23 for the production or processing of kraft pulp or paper.
  • 45. The use of claim 44, wherein said processing comprises prebleaching.
  • 46. The use of claim 44, wherein said processing comprises de-inking.
  • 47. The polypeptide of any one of claim 1 to 7 or 23 for the production or processing of kraft pulp or paper.
  • 48. The polypeptide of claim 47, wherein said processing comprises prebleaching or de-inking.
  • 49. Use of the polypeptide of any one of claim 1 to 7 or 23 for processing lignin.
  • 50. The polypeptide of any one of claim 1 to 7 or 23 for processing lignin.
  • 51. Use of the polypeptide of any one of claim 1 to 7 or 23 for producing ethanol.
  • 52. The polypeptide of any one of claim 1 to 7 or 23 for producing ethanol.
  • 53. The use of any one of claims 35, 36, 40, 41, 44 to 46, 49 and 51 in conjunction with cellulose or a cellulase.
  • 54. Use of the polypeptide of any one of claim 1 to 7 or 23 for treating textiles or dyed textiles.
  • 55. The polypeptide of any one of claim 1 to 7 or 23 for treating textiles or dyed textiles.
  • 56. Use of the polypeptide of any one of claim 1 to 7 or 23 for degrading biomass or pretreated biomass.
  • 57. The polypeptide of any one of claim 1 to 7 or 23 for degrading biomass or pretreated biomass.
PCT Information
Filing Document Filing Date Country Kind
PCT/CA2013/050434 6/7/2013 WO 00
Provisional Applications (3)
Number Date Country
61657075 Jun 2012 US
61657078 Jun 2012 US
61657082 Jun 2012 US