WO2013022594A1

WO2013022594A1 - Transcription factors for the production of cellulose degrading enzymes

Info

Publication number: WO2013022594A1
Application number: PCT/US2012/047898
Authority: WO
Inventors: Chaoguang Tian; Teresa SHOCK; N. Louise Glass; Samuel CORADETTI; James Craig
Original assignee: The Regents Of The University Of California
Priority date: 2011-07-21
Filing date: 2012-07-23
Publication date: 2013-02-14
Also published as: US20140220641A1; US9441255B2; EP2734632A1; BR112014001452A2; CN103917653A

Abstract

Provided herein are methods and compositions for increasing the production of one or more cellulases from a fungal host cell. The disclosure is based, on the surprising discovery that mis-expression of the transcriptional regulator clr-2 in a filamentous fungal cell was able to induce expression of cellulase genes under non-inducing or starvation conditions, resulting in increased secretion of cellulases from the cell. Advantageously, mis-expression of the transcription factor clr-2 in a filamentous fungal cell cultured in the absence of cellulose or cellobiose results in increased secretion of cellulases. The disclosure relates inter alia to a method of degrading cellulose-containing material, to a method of increasing the production of one or more cellulases from a fungal cell and to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding clr-2 or a related transcription factor.

Description

TRANSCRIPTION FACTORS FOR THE PRODUCTION OF CELLULOSE DEGRADING ENZYMES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No, 61/5.10,466, fi led July 21 , 201 1 , which is hereby incorporated by reference, in its entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

[G002J The conten t of the following submission on ASCII text file is incorporated herein by reference in its entirety; a computer readable form (CRF) of the Sequence Listing (file name: 677792002140SeqLisi.tx date recorded: July 23, 2012, size: 978 KB).

FIELD

10003] The disclosure relates to the degradation of cellulose. In particular, the disclosure relates to polypeptides involved in the response of a cell to cellulose, and related nucleotides and compositions. The disclosure further relates to methods and uses of polypeptides, nucleotides, and compositions thereof involved in the response of a cell to cellulose.

BACKGROUND

|0 4] Liquid fuels derived from biornass have long been studied as alternatives to fossil fuels. While the net energy yield and greenhouse gas reduction achieved with current biofuel conversion processes remains controversial biofuels produced from cellulosic feedstocks hold great potential as a source of renewable, carbon neutral liquid fuel. Current conversion processes rely heavily on enzymatic conversion of cellulose to glucose for fermentation to ethanol or other fuels. Production of these enzymes from filamentous fungi, or purchase from another party, represents a major cost in the total conversion process. Efforts to reduce this cost have been a major focus of recent public and private research on biofuel production.

[0005] The greatest advances in ceiiuiase production to date have been achieved by iterative, random mutagenesis of filamentous fungi. While this strategy has reduced the cost of enzyme production substantially, the resultant strains have hundreds of mutations, It is not clear which mutations have given rise to the desired increase in yield and which mutations are irrelevant or impair ceiiuiase production. Without a fundamental understanding of how particular mutations improve ceiiuiase yield, it will be difficult to further engineer industrial strains or transfer increased productivity to other strains of interest. A more systematic understanding of the biological process involved in cellulase production by filamentous fungi, and related compositions and methods, are needed.

BRIEF SUMMARY

[0006] In order to meet the above needs, the present disclosure provides novel methods and compositions for increasing the production of one or more cellulases from a fungal host cell. Moreover, the present disclosure is based, at least in part, on the surprising discovery that mis- expression of the transcriptional regulator clr-2 in a filamentous fungal cell was able to induce expression of cellulase genes under non-inducing or starvation conditions, resulting in increased secretion of cellulases from the cell. Advantageously, mis-expression of clr-2 in a filamentous fungal cell cultured in the absence of cellulose or cellobiose results in increased secretion of cellulases.

[0007] Accordingly, certain aspects of the present disclosure relate to a method of degrading cellulose-containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from SEQ ID NOs: 184, 185, 1 86, and 187; and b) incubating the fungal host cell and cellulose- containing material under conditions sufficient for the fungal host cell to degrade the cellulose- containing material. In certain embodiments, the transcription factor protein contains at least one additional polypeptide sequence selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least two additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least three additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains SEQ ID NOs: 184, 185, 186, and 187.

[0008] Other aspects of the present disclosure relate to a method of degrading cellulose- containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NO: 184; and b) incubating the fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade the cellulose-containing material. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 185. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 185 and 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 185 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 1 86 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 1 85, 1 86, and 1 87.

[0009] Other aspects of the present disclosure relate to a method of degrading cellulose- containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184 and 185; and b) incubating the fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade the cellulose-containing material. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 1 86 and 187.

[0010] Other aspects of the present disclosure relate to a method of degrading cellulose- containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184, 185, and 186; and b) incubating the fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade the cellulose-containing material. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 187. [0011] Other aspects of the present disclosure relate to a method of degrading cellulose- containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184, 185, 186, and 187; and b) incubating the fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade the cellulose-containing material.

[0012] In certain embodiments that may be combined with any of the preceding

embodiments, the fungal host cell is incubated under conditions sufficient for the fungal host cell to express the transcription factor protein. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell produces a greater amount of one or more cellulases than a corresponding fungal host cell lacking the at least one recombinant nucleic acid.

In certain embodiments that may be combined with any of the preceding embodiments, the cellulose-containing material contains biomass. In certain embodiments, the biomass is subjected to pretreatment prior to being contacted with the fungal host cell. In certain embodiments, the pretreatment contains one or more treatments selected from ammonia fiber expansion (AFEX), steam explosion, treatment with high temperature, treatment with high pressure, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid. In certain embodiments that may be combined with any of the preceding embodiments, the biomass contains a plant material. In certain embodiments, the plant material is selected from Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried

Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, and energy cane. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of at least one biofuel. In certain embodiments, the method further includes incubating the fungal host cell with the degraded cellulose-containing material under conditions sufficient for the fungal host cell to convert the cellulose-containing material to at least one biofuel. In certain embodiments that may be combined with any of the preceding embodiments, the biofuel is selected from ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-l -butanol, 2-methyl-l - butanol, 3 -methyl- 1-pentanol, and octanol. In certain embodiments that may be combined with any of the preceding embodiments, the degraded cellulose-containing material is cultured with a fermentative microorganism under conditions sufficient to produce at least one fermentation product from the degraded cellulose-containing material.

[0013] Other aspects of the present disclosure relate to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from SEQ ID NOs: 1 84, 185, 1 86, and 1 87; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid. In certain embodiments, the transcription factor protein contains at least one additional polypeptide sequence selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least two additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least three additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the fungal host cell is cultured in the absence of cellulose.

[0014] Other aspects of the present disclosure relate to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NO: 184; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid. In certain

embodiments, the transcription factor protein further contains SEQ ID NO: 185. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 185 and 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 185 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 186 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 185, 186, and 187. In certain embodiments, the fungal host cell is cultured in the absence of cellulose.

[0015] Other aspects of the present disclosure relate to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184 and 185; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 186 and 187. In certain embodiments, the fungal host cell is cultured in the absence of cellulose.

[0016] Other aspects of the present disclosure relate to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184, 185, and 186; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 187. In certain embodiments, the fungal host cell is cultured in the absence of cellulose.

[0017] Other aspects of the present disclosure relate to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184, 185, 186, and 187; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid. In certain embodiments, the fungal host cell is cultured in the absence of cellulose.

[0018] In certain embodiments that may be combined with any of the preceding

embodiments, the at least one recombinant nucleic acid encodes a clr-2 transcription factor protein. In certain embodiments that may be combined with any of the preceding embodiments, the at least one recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165. In certain embodiments that may be combined with any of the preceding embodiments, the at least one recombinant nucleic acid is operatively linked to a promoter selected from ccg-1, gpd-1, vvd, qa- 2, pdA, trpC, tef-1, and xlr-1.

[0019] In certain embodiments that may be combined with any of the preceding

embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor protein, where the additional transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from SEQ ID NOs: 188, 189, 190, 191, and 192. In certain embodiments, the additional transcription factor protein contains at least one additional polypeptide sequence selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments, the additional transcription factor protein contains at least two additional polypeptide sequences selected from SEQ ID NOs: 1 88, 189, 190, 191 , and 192. In certain embodiments, the additional transcription factor protein contains at least three additional polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191, and 192. In certain embodiments, the additional transcription factor protein contains at least four additional polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191, and 192. In certain embodiments, the additional transcription factor protein contains SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor protein, where the additional transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NO: 188. In certain embodiments, the additional transcription factor protein further contains SEQ ID NO: 189. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NO: 190. In certain embodiments that may be combined with any of the preceding

embodiments, the additional transcription factor further contains SEQ ID NO: 189 and 190. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NO: 191. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NOs: 189 and 191. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NOs: 190 and 191. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NO: 192. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NOs: 189 and 192. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NOs: 190 and 192. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NOs: 191 and 192. In certain embodiments that may be combined with any of the preceding embodiments, the additional transcription factor further contains SEQ ID NO: 189, 190, 191 , and 192. In certain embodiments that may be combined with any of the preceding embodiments, the at least one additional recombinant nucleic acid encodes a clr-1 transcription factor protein. In certain embodiments that may be combined with any of the preceding embodiments, the at least one additional recombinant nucleic acid encoding the additional transcription factor is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183. In certain embodiments that may be combined with any of the preceding embodiments, the at least one additional recombinant nucleic acid is operatively linked to a promoter selected from ccg-1, gpd-1, vvd, qa-2, pdA, trpC, tef-1, and xlr- 1.

[0020] In certain embodiments that may be combined with any of the preceding

embodiments, the fungal host cell further contains at least one recombinant nucleic acid encoding a hemicellulase. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell is selected from Neurospora crassa, Metarhizium anisopliae, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotionim,

Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile

(Myceliophthora thermophila) , Thielavia terres/ra-thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris,

Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum) , Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), and Chrysosporium lucknowense.

[0021] Other aspects of the present disclosure relate to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least one additional polypeptide sequence selected from SEQ ID NOs: 184, 185, 186, and 187. In certain

embodiments, the transcription factor protein contains at least two additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains at least three additional polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the transcription factor protein contains SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-2 transcription factor protein. In certain embodiments, the at least one recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor, where the additional transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-1 transcription factor protein. In certain embodiments, the at least one additional recombinant nucleic acid encoding the additional transcription factor protein is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183. [0022] Other aspects of the present disclosure relate to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NO: 184. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 185. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 185 and 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 185 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NOs: 186 and 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 185, 186, and 187. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr- 2 transcription factor protein. In certain embodiments, the at least one recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor, where the additional transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a

PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr- 1 transcription factor protein. In certain embodiments, the at least one additional recombinant nucleic acid encoding the additional transcription factor protein is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183.

[0023] Other aspects of the present disclosure relate to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184 and 185. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 186. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 187. In certain embodiments that may be combined with any of the preceding embodiments, the transcription factor further contains SEQ ID NO: 186 and 187. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-2 transcription factor protein. In certain embodiments, the at least one recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor, where the additional transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 1 89, 190, 191 , and 192. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-1 transcription factor protein. In certain embodiments, the at least one additional recombinant nucleic acid encoding the additional transcription factor protein is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183.

[0024] Other aspects of the present disclosure relate to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and SEQ ID NOs: 184, 185, and 186. In certain embodiments, the transcription factor protein further contains SEQ ID NO: 187. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-2 transcription factor protein. In certain embodiments, the at least one recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165. In certain embodiments that may be combined with any of the preceding embodiments, the fungal host cell further contains at least one additional recombinant nucleic acid encoding an additional transcription factor, where the additional transcription factor protein contains a zinc(2)- cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments, the at least one additional recombinant nucleic acid encodes a clr-1 transcription factor protein. In certain embodiments, the at least one additional recombinant nucleic acid encoding the additional transcription factor protein is SEQ ID NO: 2, SEQ ID NO: 119, or SEQ ID NO: 183. [0025] In some embodiments, provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein.

[0026] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, where the recombinant nucleic acid is SEQ ID NO: 5 or SEQ ID NO: 165.

[0027] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, where the cell further contains at least one additional recombinant nucleic acid encoding a clr- 1 transcription factor protein.

[0028] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, and where the recombinant nucleic acid encoding a clr-1 protein is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183.

[0029] Further provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr- 1 transcription factor protein, where the recombinant nucleic acid encoding a clr-2 protein is SEQ ID NO: 5 or SEQ ID NO: 165, and the recombinant nucleic acid encoding a clr-1 protein is SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183.

[0030] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, where the cell further contains one or more additional recombinant nucleic acids encoding a hemicellulase.

[0031] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, where the cell further contains one or more recombinant nucleic acids encoding a hemicellulase.

[0032] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, where the host cell is selected from

Nenrospora crassa, Metarhizium anisopliae, Gibberella zeae, Nectria haematococca,

Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaelomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila) , Thielavia terrestris-t ermophWi^'c, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

[0033] Also provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, where the host cell is selected from

Neurospora crassa, Metarhizium anisopliae, Gibberella zeae, Nectria haematococca,

Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botiyotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila), Thielavia terres/ra-thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

[0034] In some embodiments, provided herein is a method of increasing the growth of a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein in media under conditions sufficient to support the expression of said recombinant nucleic acid, where the host cell grows at a faster rate than a corresponding host cell lacking said recombinant nucleic acid. [0035] In some embodiments, provided herein is a method of increasing the growth of a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein in media under conditions sufficient to support the expression of the recombinant nucleic acids, where the host cell grows at a faster rate than a corresponding host cell lacking said recombinant nucleic acids.

[0036] In some embodiments, provided herein is a method of increasing the production of cellulases from a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein in growth media under conditions sufficient to support the expression of said recombinant nucleic acid, where the host cell produces a greater amount of cellulases than a corresponding host cell lacking said recombinant nucleic acid.

[0037] In some embodiments, provided herein is method of increasing the production of cellulases from a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein in growth media under conditions sufficient to support the expression of the recombinant nucleic acids, where the host cell produces a greater amount of cellulases than a corresponding host cell lacking said recombinant nucleic acids.

[0038] Also provided herein is a method of increasing the production of cellulases from a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein in growth media that does not contain cellulose under conditions sufficient to support the expression of said recombinant nucleic acid, where the host cell produces a greater amount of cellulases than a corresponding host cell lacking said recombinant nucleic acid.

[0039] Also provided herein is a method of increasing the production of cellulases from a fungal cell, the method including incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein in growth media that does not contain cellulose under conditions sufficient to support the expression of said recombinant nucleic acids, where the host cell produces a greater amount of cellulases than a corresponding host cell lacking said recombinant nucleic acids.

[0040] In some embodiments, provided herein is a method of preparing one or more cellulases, the method including: a) incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein in media under conditions sufficient to support the expression of said recombinant nucleic acid, and b) collecting one or more cellulases from said media and/or said fungal host cell.

[0041] In some embodiments, provided herein is a method of preparing one or more cellulases, the method including: a) incubating a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein in media under conditions sufficient to support the expression of said recombinant nucleic acids, and b) collecting one or more cellulases from said media and/or said fungal host cell.

[0042] Further provided herein is a method of degrading a cellulose-containing material, the method including: a) contacting the cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, and b) incubating the fungal host cell and cellulose-containing material under conditions that support cellulose degradation.

[0043] Also provided herein is a method of degrading a cellulose-containing material, the method including: a) contacting the cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, and b) incubating the fungal host cell and cellulose-containing material under conditions that support cellulose degradation.

[0044] Further provided herein is a method of converting a cellulose-containing material to fermentation product, the method including: a) contacting the cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product. [0045] Also provided herein is a method of converting a cellulose-containing material to fermentation product, the method including: a) contacting the cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr- 1 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0046] Also provided herein is a method of converting biomass to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0047] Also provided herein is a method of converting biomass to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0048] Also provided herein is a method of converting biomass to fermentation product, the method including: a) pretreating the biomass, b) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and c) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0049] Also provided herein is a method of converting biomass to fermentation product, the method including: a) pretreating the biomass, b) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, to yield a sugar solution, and c) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0050] Also provided herein is a method of converting biomass to fermentation product, the method including: a) pretreating the biomass by a method that includes one or more of ammonia fiber expansion (AFEX), steam explosion, treatment with high temperature, treatment with high pressure, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid, b) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and c) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0051] Also provided herein is a method of converting biomass to fermentation product, the method including: a) pretreating the biomass by a method that includes one or more of ammonia fiber expansion (AFEX), steam explosion, treatment with high temperature, treatment with high pressure, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid, b) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, to yield a sugar solution, and c) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0052] Additionally provided herein is a method of converting a plant material to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0053] Additionally provided herein is a method of converting a plant material to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0054] Also provided herein is a method of converting a plant material selected from Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, and energy cane to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0055] Also provided herein is a method of converting a plant material selected from Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, and energy cane to fermentation product, the method including: a) contacting the biomass with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr- 1 transcription factor protein, to yield a sugar solution, and b) culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0056] Further provided herein is a method of reducing the viscosity of a pretreated biomass material, the method including contacting the pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein, to yield a pretreated biomass material having reduced viscosity.

[0057] Further provided herein is a method of reducing the viscosity of a pretreated biomass material, the method including contacting the pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein and at least one additional recombinant nucleic acid encoding a clr- 1 transcription factor protein, to yield a pretreated biomass material having reduced viscosity.

[0058] In some embodiments, provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of the clr-1 and clr-2 proteins, as compared to the expression of the clr- 1 and clr-2 proteins in a corresponding fungal cell lacking said modifications.

[0059] Also provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, and where the modifications are caused by RNAi, antisense RNA, T-DNA insertion, transposon insertion, insertional mutagenesis, site-directed mutagenesis, partial deletion of the gene, or complete deletion of the gene.

[0060] Also provided herein is a non-naturally occurring Neurospora cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said

modifications.

[0061] Also provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr- 1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, and where the cell further contains a recombinant nucleic acid encoding a polypeptide involved in cellulose metabolism.

[0062] Also provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, and where the cell further contains a recombinant nucleic acid encoding a cellulase.

[0063] In some embodiments, provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides and at least one gene orthologous to the

Neurospora crassa gene SEQ ID NO: 5, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification. [0064] In some embodiments, provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 , as compared with a corresponding cell lacking said modifications, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications.

[0065] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification, and where the modification(s) are caused by RNAi, antisense RNA, T-DNA insertion, transposon insertion, insertional mutagenesis, site-directed mutagenesis, partial deletion of the gene, or complete deletion of the gene.

[0066] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 , as compared with a corresponding cell lacking said modifications, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications, and where the modification(s) are caused by RNAi, antisense RNA, T-DNA insertion, transposon insertion, insertional mutagenesis, site-directed mutagenesis, partial deletion of the gene, or complete deletion of the gene.

[0067] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification, and where the cell contains one or more RNAi-inducing vectors, where the one or more vectors generate RNAi against one or more genes orthologous to the Neurospora crassa gene SEQ ID NO: 5.

[0068] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 , as compared with a corresponding cell lacking said modifications, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications, and where the cell contains one or more RNAi-inducing vectors, where the one or more vectors generate RNAi against one or more genes orthologous to the Neurospora crassa gene SEQ ID NO: 5 or SEQ ID NO: 2.

[0069] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and a recombinant nucleic acid encoding a polypeptide involved in cellulose metabolism, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification.

[0070] Also provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, and a recombinant nucleic acid encoding a polypeptide involved in cellulose metabolism, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 , as compared with a corresponding cell lacking said modifications, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications.

[0071] Additionally provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and a recombinant nucleic acid encoding a cellulase, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification.

[0072] Additionally provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, and a recombinant nucleic acid encoding a cellulase, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2, as compared with a corresponding cell lacking said modifications, and, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications.

[0073] Further provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5, as compared with a corresponding cell lacking said modification, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modification, and where the host cell is selected from Metarhizium anisopliae, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-alrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila) , Thielavia /erre^ra-thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

[0074] Further provided herein is a non-Neurospora cell, containing DNA encoding one or more cellulase polypeptides, at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 5, and at least one gene orthologous to the Neurospora crassa gene SEQ ID NO: 2, where the cell contains at least one modification causing reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one modification causing reduced expression of one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 , as compared with a corresponding cell lacking said modifications, where the reduced expression of at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 5 and at least one of said gene(s) orthologous to the Neurospora crassa gene SEQ ID NO: 2 causes reduced expression of one or more of said cellulase polypeptides, as compared with expression of said cellulase polypeptides in a corresponding cell lacking said modifications, and where the host cell is selected from Metarhizium anisopliae, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila), Thielavia terrestris- thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha,

Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

[0075] In another embodiment, provided herein is a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

[0076] In another embodiment, provided herein is a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein and a recombinant nucleic acid encoding a clr- 1 transcription factor protein, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

[0077] In another embodiment, provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

[0078] Also provided herein is a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel, and where the biofuel is selected from ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-l-butanol, 2-methyl-l -butanol, 3 -methyl- 1-pentanol, and octanol.

[0079] Also provided herein is a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein and a recombinant nucleic acid encoding a clr-1 transcription factor protein, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel, and where the biofuel is selected from ethanol, n-propanol, n-butanol, iso-butanol, 3- methyl-l-butanol, 2-methyl-l-butanol, 3 -methyl- 1 -pentanol, and octanol.

[0080] Also provided herein is a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr- 1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said

modifications, where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel, and where the biofuel is selected from ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-l-butanol, 2- methyl-l-butanol, 3 -methyl- 1 -pentanol, and octanol.

[0081] Further provided herein is a method of converting a cellulose-containing material to fermentation product, the method including contacting the cellulose-containing material with a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein, and where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

[0082] Further provided herein is a method of converting a cellulose-containing material to fermentation product, the method including contacting the cellulose-containing material with a fungal host cell containing a recombinant nucleic acid encoding a clr-2 transcription factor protein and a recombinant nucleic acid encoding a clr-1 transcription factor protein, and where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

[0083] Further provided herein is a method of converting a cellulose-containing material to fermentation product, the method including contacting the cellulose-containing material with a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, and where the cell further contains one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of a biofuel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

[0085] Figure 1 depicts expression patterns for secreted enzymes after media shift. Figure 1A depicts typical expression patterns for secreted enzymes after shift to no carbon or a new carbon source. Figure IB depicts message abundance for 16 predicted Neurospora crassa cellulases after cultures are shifted to no carbon or a new carbon source. Figure 1C depicts message abundance for 12 predicted N. crassa hemicelluses after cultures are shifted to a no carbon or a new carbon source. Abundances are given as fragments per kilobase of exon length per million reads fragments (FPKM) as calculated by Cufflinks.

[0086] Figure 2A depicts transcript abundance for the full Neurospora crassa (N crassa) genome as compared between cellulose and sucrose cultures at 1 hour after transfer. Figure 2B depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between sucrose and no-carbon at 1 hour. Figure 2C depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and no-carbon at 1 hour. Figure 2D depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and sucrose at 4 hours. Figure 2E depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between sucrose and no-carbon at 4 hours. Figure 2F depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and no-carbon at 4 hours. Log₂ fold change is plotted against maximum abundance. For plotting purposes genes are given a minimum count of 1 FPKM in all conditions. Light-gray points are not statistically different by the model employed by Cuffdiff. Medium-gray points are statistically different, but not consistently different by a factor of 2 or more. Dark-gray / black points are statistically different and consistently different by 2-fold.

[0087] Figure 3 depicts a comparison of differentially expressed genes from RNAseq and microarray data. Both Cellulose (CMM) vs. no-carbon (NC) and Cellulose vs. sucrose (SMM) conditions are compared. Figure 3A depicts gene sets of differentially expressed genes from CMM vs. NC (RNAseq data, purple circle), and differentially expressed genes from CMM vs. SMM (RNAseq data, blue circle). A third gene set (Microarray data, red circle), includes differentially expressed genes from cultures grown on CMM for 30 hours vs SMM for 16hrs, Tian et al., Proc Nat Acad Sci USA, 106: 22157-22162 (2009). Each of the 3 sets of

differentially expressed genes includes both the up-regulated and down-regulated genes. Figure 3B depicts a comparison of the RNAseq derived differentially expressed gene lists from Figure 3A and separates them into up-regulated and down-regulated gene sets (The microarray data was not included in this analysis). White arrows pointing upward indicate genes that are up-regulated and downward pointing arrows indicate down-regulated genes.

[0088] Figure 4 depicts growth and enzyme secretion of deletion strains for cdr-1 (clr-J) and cdr-2 (clr-2). Figure 4A depicts growth of wild type and deletion strains in 5 ml tubes with SMM, XMM or CMM. Figure 4B depicts growth on a layer of saturated cellulose minimal medium. Figure 4C depicts an SDS-PAGE gel of culture supernatants from SMM cultures (16 hr) transferred to CMM or XMM and incubated for 24 hrs. In the SDS-PAGE gel, lane 1 shows the protein ladder; lane 2 shows the results of the wild-type strain grown on sucrose; lane 3 shows the results of the Aclr-l deletion strain grown on sucrose; lane 4 shows the results of the Aclr-2 deletion strain grown on sucrose; lane 5 shows the results of the wild-type strain grown on xylan; lane 6 shows the results of the Aclr-l deletion strain grown on xylan; lane 7 shows the results of the Aclr-2 deletion strain grown on xylan; lane 8 shows the results of the wild-type strain grown on Avicel^®; lane 9 shows the results of the Aclr-l deletion strain grown on Avicel^®; and lane 10 shows the results of the Aclr-2 deletion strain grown on Avicel^®. Figure 4D depicts total cellulase activity as measured by glucose release from cellulose (Tian et al., Proc Nat Acad Sci USA, 106: 22157-22162 (2009)) in supernatants from 16 hr SMM cultures transferred to either CMM or XMM for 24 hrs. Figure 4E depicts total xylanase activity as measured by reducing sugars released from xylan from CMM or XMM cultures from Figure 4D. Figure 4F depicts total protein as measured by the Bradford assay in CMM or XMM cultures from Figure 4D.

[0089] Figure 5A depicts the domain architecture of cdr-1 (clr-1) and cdr-2 (clr-2) showing PFAM domains that are conserved among Zn(2) Cys(6) binuclear cluster transcription factors. Figure 5B depicts the construct design for natively tagged cdr-l-GFP tagging and a mis- expression cdr-1 construct (under regulation of the ccg-1 promoter). Figure 5C depicts expression profiles of cdr-1 and cdr-2 following shift of a SMM-grown culture to CMM. Figure 5D depicts FPKMs derived from cdr-1 and cdr-2 in a wild type N. crassa versus a cdr-1 or cdr-2 mutant. Note that expression of cdr-2 is dependent upon the presence of functional cdr-1, while expression of cdr-1 is similar to wild type in a cdr-2 mutant. Figure 5E depicts the nuclear localization of natively GFP tagged CDR-1 (CLR-1). Figure 5F depicts the relative expression of cbh-1 (NCU07340) and cdr-2 in the ccgl::cdr-l strain on CMM versus SMM indicates that mis-expression of cdr-1 has no effect on expression levels of either cbh-1 or cdr-2.

[0090] Figure 6 depicts maximum likelihood phylogenetic trees of cdr-1 (clr-1) and cdr-2 (clr-2). Figure 6A depicts cdr-1. Figure 6B depicts cdr-2.

[0091] Figure 7 depicts altered expression profiles in cdr (clr) deletion mutants. Figure 7A depicts transcript abundance of predicted cellulase genes in wild type and cdr mutant strains at 4 hrs after transfer to CMM. Figure 7B depicts expression profiles of predicted hemicellulase genes in wild type and cdr mutant strains at 4 hrs after transfer to CMM. Figure 7C depicts global expression in Acdr-1 (Aclr-1) as compared to wild type after transfer to CMM for 4 hrs. Figure 7D depicts global expression in Acdr-2 (Aclr-2) as compared to wild type after transfer to CMM for 4 hrs. Figure 7E depicts hierarchical clustering of FPKM at 4 hrs after transfer to CMM for genes identified as differentially expressed in clr mutants and/or in the wild type cellulose to no-carbon comparison. Figure 7F depicts major classes of genes in the clusters from Figure 7E. Figure 7G depicts FPKM of selected cellulases and hemicellulases. Predicted hemicellulases exhibiting cellulase-like expression patterns are regulated by cdr-1 and cdr-2.

[0092] Figure 8 depicts a non-limiting model for cellulase regulation by cdr-1 (clr-1) and cdr-2 (clr-2). (1) Glucose repression is released. (2) Scout cellulases and hemicellulases degrade plant cell wall material, releasing signal molecules. (3, 4) Signal cascade activates CDR-1 (CLR-1), driving further expression of cdr-1 followed by cdr-2. (5) CDR-2 (CLR-2) and possibly CDR-1 drives expression of the cellulases and some hemicellulases. (6) Cellulases and hemicellulases release more signal molecules, perpetuating the cycle.

[0093] Figure 9 depicts phylogenetic trees based on Bayesian inference. Figure 9A depicts a cdr-1 (clr-l) tree. Figure 9B depicts a cdr-2 (clr-2) tree.

[0094] Figure 10 depicts transcript abundance of cbh-1 and cdt-2 in triple beta-glucosidase deletion mutants (ABG) with or without deletion of clr-l or clr-2 four hours after shift to 0.2% cellobiose.

[0095] Figure 11 A depicts cellulase activity in culture supernatants as measured by cellobiose release from Avicel®. Cultures were grown 24 hours on sucrose then transferred to fresh media. Figure 11B depicts transcription of cbh-1 as a function of clr-l abundance. All measurements are by RT-PCR 4 hours after media shift from sucrose cultures.

[0096] Figure 12A depicts transcript abundance of cbh-1 relative to clr-2 in N. crassa strains 4 hours after shift from sucrose media. Figure 12B depicts CMCase activity in WT and clr-2 mis-expression strain supernatants after growth in sucrose or Avicel®. Figure 12C depicts CMCase activity of clr-2 mis-expression strains after a sucrose grown culture was shifted to fresh media with 2% sucrose or 2% Avicel®. Figure 12D depicts secreted protein in culture supernatants from clr-2 mis-expression strains after a sucrose grown culture was shifted to fresh media with 2% sucrose or 2% Avicel®.

[0097] Figure 13A depicts an SDS-PAGE gel of culture supernatants from WT and clr-2 mis-expression strains. Figure 13B depicts transcript abundance (RNAseq) of selected cellulase genes in WT N. crassa, deletion strains for clr-l and clr-2 after transfer to Avicel®; and WT and clr-2 mis-expression strains after transfer to no carbon.

[0098] Figure 14 depicts hierarchical clustering of the N. crassa Avicel® regulon by FPKM in alternative inducing conditions and clr mutants.

[0099] Figure 15 depicts a Western blot (anti-V5 antibody) of tagged and untagged clr-l (NCU07705) in N. crassa lysates 4 hours after media shift to various carbon sources. Sue refers to sucrose, Avi refers to Avicel®, NC refers to no carbon, Cel refers to cellobiose, Xa refers to xylan, and Xo refers to xylose. The predicted size of the V5 tagged CLR-1 is -80 kDa. Equal total protein concentrations were loaded per lane. [0100] Figure 16A depicts a Venn Diagram comparing the clr-1/2 ChiPseq regulons to the cellulose response RNAseq regulon. Figure 16B depicts a graphical representation of the CLR- 1 ChlP-Seq. The grey peaks represent the relative number of reads mapping to several sites within the promoter regions of clr-1 (NCU07705) and clr-2 (NCU08042). Figure 16C depicts CLR-1 and CLR-2 ChlP-Seq as well as a 4 hour Avicel® RNA-Seq mapped to the genome. The figure shows the typical ChIP binding pattern of CLR-1 and CLR-2 when they regulate the same gene. CLR-1 and CLR-2 bind to the promoter ofxlr-1 in nearly identical places.

[0101] Figure 17 depicts the phenotype of A. nidulans clr deletion strains AclrA and AclrB. Figure 17A depicts the enzyme activity of culture supernatants from AclrA and AclrB mutants grown on glucose and then shifted to Avicel® media. Figure 17B depicts the total protein in supernatants of cultures grown on Avicel®. Figure 17C depicts mycelial dry weights from WT and clr mutants from cultures on glucose and cellobiose (0.5% wt/vol). Figure 17D depicts induction of selected cellulase genes in WT and the clr mutants following an 8 hr shift to Avicel®, by quantitative RT-PCR. Statistical significance by one tailed, unequal variance t-test. *P < 0.05, **P < 0.01 , ***P < 0.001. Figure 17E depicts the expression of clrA and clrB in Aspergillus nidulans AclrA and AclrB mutants after the cultures were exposed to Avicel®. The culture were pre-grown in glucose media for 17 hrs at 37°C and then shifted to Avicel® media.

[0102] Figure 18A depicts the expression of the clrB gene in the clrB mis-expression strain. 0 hrs on glucose refers to the time just before shifting the culture grown 17 hr on glucose to media with other carbon source. Figure 18B depicts the expression of the cbhD gene in the clrB mis-expression strain. 0 hrs on glucose refers to the time just before shifting the culture grown 17 hr on glucose to media with other carbon source. Figure 18C depicts the growth and CMCase activity of clrB mis-expression strain grown on cellobiose for 48 hrs.

[0103] Figure 19 depicts growth of an N. crassa clrA mis-expression strain and an N. crassa clrB mis-expression strain on cellobiose and Avicel®. Figure 19A depicts biomass

accumulation of the clrA mis-expression strain on cellobiose. Figure 19B depicts growth of the clrA mis-expression strain on Avicel®. Figure 19C depicts biomass accumulation of the clrB mis-expression strain on cellobiose. Figure 19D depicts growth of the clrB mis-expression strain on Avicel®. [0104] Figure 20A depicts the Clr-1 DNA-binding motif as predicted from chromatin immunoprecipitation (ChIP) peaks. Figure 20B depicts the Clr-2 DNA-binding motif as predicted from chromatin immunoprecipitation (ChIP) peaks.

[0105] Figure 21 depicts an amino acid sequence alignment of N. crassa clr-1 with 22 clr- 1 homologs showing conserved motifs. The conserved PFAM04082 transcription factor domain is boxed in dashes. The sequence alignment included the following sequences:

GibbereIla_zeae_PH-l (SEQ ID NO: 193), Nectria_haematococca_mpVI_77-13-4 (SEQ ID NO: 194), NCU07705 (SEQ ID NO: 2), Neurospora_tetrasperma_^FGSC__2508 (SEQ ID NO: 195), Sordaria_macrospora_k-hell (SEQ ID NO: 196), Chaetomium_globosum_CBSJ 48.51 (SEQ ID NO: 197), Podospora_anserina_S_mat+ (SEQ ID NO: 198), Verticillium_albo-atrum_VaMs. l 02 (SEQ ID NO: 199), Glomerella_graminicola_M 1.001 (SEQ ID NO: 200),

Metarhizium_anisopliae_ARSEF_23 (SEQ ID NO: 201), Botryotinia_fuckeliana_B05.10 (SEQ ID NO: 202), Sclerotinia_sclerotiorum_1980 (SEQ ID NO: 203),

Grosmannia_„clavigera_kwl407 (SEQ ID NO: 204), AN5808 (SEQ ID NO: 205),

Aspergillus_fumigatus_Af293 (SEQ ID NO: 206), Aspergillus_oryzae_RIB40 (SEQ ID NO: 207), Penicillium_chrysogenum_Wisconsin_54-1255 (SEQ ID NO: 208), Aspergillus niger (SEQ ID NO: 209), Pyrenophora_teres_^f.jeres_0-1 (SEQ ID NO: 210),

Leptosphaeria_maculans_JN3 (SEQ ID NO: 21 1), Talaromyces_stipitatus_ATCC_10500 (SEQ ID NO: 212), NCU00808 (SEQ ID NO: 213), and Trichoderma_reesei_clr-l_protein (SEQ ID NO: 182).

[0106] Figure 22 depicts an amino acid sequence alignment of N. crassa clr-1 with 21 clr-2 homologs showing conserved motifs. The conserved PFAM04082 transcription factor domain is boxed in dashes. The sequence alignment included the following sequences: ΑΝ6832 (SEQ ID NO: 214), Penicilliumjnarneffei_ATCC_18224 (SEQ ID NO: 215),

Talaromyces_stipitatus_ATCC_10500 (SEQ ID NO: 216), AN3369 (SEQ ID NO: 217), Aspergillus jtiger_CBS_513.88 (SEQ ID NO: 218), Aspergillus_oryzae_RIB40 (SEQ ID NO: 219), Penicillium_chrysogenum_Wisconsin_54-1255 (SEQ ID NO: 220),

Coccidioides immitis RS (SEQ ID NO: 221), Coccidioides_posadasii_C735_delta_SOWgp (SEQ ID NO: 222), NCU08042 (SEQ ID NO: 4), Neurospora_tetrasperma_FGSC_2508 (SEQ ID NO: 223), Sordaria_macrospora_k-hell (SEQ ID NO: 224), Podospora_anserina_S_mat+ (SEQ ID NO: 225), Glomerella_graminicola_M 1.001 (SEQ ID NO: 226),

Magnaporthe_oryzae_70-15 (SEQ ID NO: 227), Nectria_haematococca_mpVI_77-13-4 (SEQ ID NO: 228), Trichoderma_reesei (SEQ ID NO: 229), Verticillium_albo-atrum_VaMs. l02 (SEQ ID NO: 230), Pyrenophora_tritici-repentis__Pt-lC-BFP (SEQ ID NO: 231),

Pyrenophora_teres_f._teres_0-1 (SEQ ID NO: 232), Leptosphaeria_maculans_JN3 (SEQ ID NO: 233), and NCU07007 (SEQ ID NO: 234).

DETAILED DESCRIPTION

[0107] Provided herein are polypeptides involved in the response of cells to cellulose.

Further provided herein are nucleic acids encoding polypeptides involved in the response of cells to cellulose. Also provide herein are host cells containing recombinant nucleic acids encoding polypeptides involved in the response of cells to cellulose, and host cells containing recombinant polypeptides involved in the response of cells to cellulose. In some aspects, provided herein are the polypeptides clr-1 and clr-2, and nucleic acids encoding clr-1 and clr-2 polypeptides.

[0108] Further provided herein are methods for use of clr-1 and clr-2 polypeptides, methods for use of nucleic acids encoding clr-1 and clr-2 polypeptides, and methods for use of host cells containing recombinant clr-1 and/or clr-2 polypeptides or nucleic acids encoding clr-1 and/or clr- 2 polypeptides. In some aspects, clr-1 and clr-2 promote the expression of cellulases and other genes in response to cellulose. Accordingly, in some aspects, the expression of recombinant clr- 1 and/or clr-2 in a host cell increases the growth rate of a host cell on media containing cellulose, increases the production of cellulases from the host cell, and/or increases the rate of cellulose degradation by the host cell.

[0109] In addition, provided herein are cells that naturally produce clr-1 and/or clr-2 polypeptides, which are modified to have reduced expression of clr-1 and/or clr-2. Cells which naturally produce clr-1 and/or clr-2 polypeptides, but which are modified to have reduced expression of clr-1 and/or clr-2 may be used, for example to study cellulases and the response of cells to cellulose.

[0110] As used herein the terms "cdr-1 " and "clr-1" are used interchangeably and refer to polypeptides or genes encoding polypeptides that function as transcription factors that regulate the transcription of various genes in a fungal cell in response to the exposure of the cell to cellulose. One non-limiting example of a clr-1 encoding gene is the N. crassa gene NCU07705.

[0111] As used herein the terms "cdr-2" and "clr-2" are used interchangeably and refer to polypeptides or genes encoding polypeptides that function as transcription factors that regulate the transcription of various genes in a fungal cell in response to the exposure of the cell to cellulose. One non-limiting example of a clr-2 encoding gene is the N. crassa gene NCU08042.

[0112] Accordingly, in certain aspects the present disclosure relates to a method of degrading cellulose-containing material, by: a) contacting cellulose-containing material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187; and b) incubating the fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade the cellulose-containing material.

[0113] Other aspects the present disclosure relates to a method of increasing the production of one or more cellulases from a fungal cell, by: (a) providing a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, where the

transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a

PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187; and (b) culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid.

[0114] Other aspects the present disclosure relates to a method of reducing the viscosity of a pretreated biomass material, by contacting pretreated biomass material with a fungal host cell containing at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, where the transcription factor protein contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187.

Polypeptides of the Disclosure

[0115] The present disclosure relates to polypeptides that are involved in the transcription of genes related to cellulose metabolism. In some aspects, the disclosure relates to clr-1 polypeptides. In some aspects, the disclosure relates to clr-2 polypeptides. [0116] As used herein, a "polypeptide" is an amino acid sequence including a plurality of consecutive polymerized amino acid residues (e.g., at least about 15 consecutive polymerized amino acid residues). As used herein, "polypeptide" refers to an amino acid sequence, oligopeptide, peptide, protein, or portions thereof, and the terms "polypeptide" and "protein" are used interchangeably.

Clr-1

[0117] In some aspects, the present disclosure relates to clr-1 polypeptides. Clr-1 polypeptides function as transcription factors that regulate the transcription of various genes in a fungal cell in response to the exposure of the cell to cellulose. In some aspects, the expression of a gene is increased in response to clr-1 expression. In some aspects, the expression of a gene is decreased in response to clr-1 expression.

[0118] Clr-1 is a member of the fungal specific zinc binuclear cluster superfamily, which is large, diverse superfamily of fungal-specific transcriptional regulators. Examples of transcription factors in this superfamily include gal-4, ace-1 , and xlnR (xyr-1) (Strieker et al., App. Micro. Biotech., 78: 21 1 -220 (2008)). Clr-2 is also a member of this superfamily.

[0119] Members of this polypeptide superfamily typically contain two conserved domains: A) a zinc(2)-cysteine(6) binuclear cluster PFAM00172 domain, which coordinates binding of the polypeptide to the DNA, and B) a central domain, which roughly corresponds to what is known as the "middle homology region" (Campbell RN, Biochemical J., 414: 177-187, (2008)), a conserved domain in zinc finger transcription factors. In clr-1 , the conserved central domain has the fungal-specific transcription factor domain PFAM04082.

[0120] As used herein, a "zinc(2)-cysteine(6) binuclear cluster domain" refers to the conserved DNA-binding domain of the fungal specific zinc binuclear cluster superfamily, typified by Saccharomyces cerevisiae Gal4, that contains a binuclear zinc cluster in which two zinc ions are bound by six cysteine residues (PFAM00172). Clr-1 polypeptides of the present disclosure, and homologs thereof, contain a "zinc(2)-cysteine(6) binuclear cluster domain" that includes the following conserved sequence: C-E-V-C-R-S-R-K-S-R-C-D-G-T-K-P-K-C- -L-C- T-E-L-G-A-E-C-I-Y-R-E (SEQ ID NO: 235) (Fig. 21). Clr-2 polypeptides of the present disclosure, and homologs thereof, contain a "zinc(2)-cysteine(6) binuclear cluster domain" that includes the following conserved sequence: C-A-E-C-R-R-R-K-I-R-C-D-G-E-Q-PC-G-Q-C-X- W-Y-X-K-P-K-R-C-F-Y-R-V-X-P-S-R-K (SEQ ID NO: 236), where X can be any amino acid residue (Fig. 22).

[0121] As used herein, a "PFAM04082 transcription factor domain" refers to a fungal- specific transcription factor domain that is associated with a zinc finger or zinc binuclear transcription factor domain. Clr-1 polypeptides of the present disclosure, and homologs thereof, contain a "PFAM04082 transcription factor domain" that includes the following conserved sequence: I-E-A-Y-F-E-R-V-N-V-W-Y-A-C-V-N-P-Y-T-W-R-S-H-Y-R-T-A-L-S-N-G-F-R-E- G-P-E-S-C-I-V-L-L-V-L-A-L-G-Q-A-S-L-R-G-S-l-S-R-I-V-P-X-E-D-P-P-G-L-Q-Y-F-T-A-A- W-X-L-L-P-G-M-M-T-X-N-S-V-L-A-A-Q-C-H-L-L-A-A-A-Y-L-F-Y-L-V-R-P-L-E-A-W-N-L- L-C-T-T-S-T-K-L-Q-L-L-L-M-A-P-N-R-V-P-P-X-Q-R-E-L-S-E-R-I-Y-W-N-A-L-L-F-E-S-D- L-L-A-E-L-D-L-P-H-S-G-I-V-Q-F-E-E-N-V-G-L-P-G-G-F-E-G-E-E-D-E-X-D-E-E-A-D-X-D- Q-E-I-A-X-V-T-A-V-G-R-D-E-L-W-Y-F-L-A-E-I-A-L-R-R-L-L-N-R-V-S-Q-L-I-Y-S-K-D-T- P-Y-S-K-G-P-S-M-A-S-T-T-S-L-E-P-I-V-A-E-L-D-F-Q-L-T-Q-W-Y-E (SEQ ID NO: 237), where X can be any amino acid residue (Fig. 21). Clr-2 polypeptides of the present disclosure, and homologs thereof, contain a "PFAM04082 transcription factor domain" that includes the following conserved sequence: I-D-A-Y-F-K-R-V-H-X-F-X-P-M-L-D-E-X-T-F-R-A-T-Y-L-E- G-Q-R-K-D-A-P-W-L-A-L-L-N-M-V-F-A-L-G-S-I-A-A-M-K-S-D-D-Y-N-H-X-X-Y-Y-N-R- A-M-E-H-L-X-L-D-S-F-G-S-S-H-X-E-T-V-Q-A-L-A-L-M-G-G-Y-Y-L-H-Y-I-N-R-P-N-X-A- N-A-L-M-G-A-A-L-R-M-A-S-A-L-G-L-FI-R-E-S-L-A-Q-X-X-A-S-S-Q-K-G-V-N-X-S-D-X-A- S-A-E-T-R-R-R-T-W-W-S-L-F-C-L-D-T-W-A-T-T-T-L-G-R-P-S-X-G-R-W-G (SEQ ID NO: 238), where X can be any amino acid residue (Fig. 22).

[0122] Accordingly, in certain embodiments, clr-1 polypeptides of the present disclosure have a zinc(2)-cysteine(6) binuclear cluster domain having the following conserved sequence: C-

E-V-C-R-S-R-K-S-R-C-D-G-T-K-P-K-C-K-L-C-T-E-L-G-A-E-C-I-Y-R-E (SEQ ID NO: 235); and a PFAM04082 transcription factor domain having the following conserved sequence: I-E-A-

Y-F-E-R-V-N-V-W-Y-A-C-V-N-P-Y-T-W-R-S-H-Y-R-T-A-L-S-N-G-F-R-E-G-P-E-S-C-I-V-L-

L-V-L-A-L-G-Q-A-S-L-R-G-S-I-S-R-I-V-P-X-E-D-P-P-G-L-Q-Y-F-T-A-A-W-X-L-L-P-G-M-

M-T-X-N-S-V-L-A-A-Q-C-H-L-L-A-A-A-Y-L-F-Y-L-V-R-P-L-E-A-W-N-L-L-C-T-T-S-T-K-

L-Q-L-L-L-M-A-P-N-R-V-P-P-X-Q-R-E-L-S-E-R-I-Y-W-N-A-L-L-F-E-S-D-L-L-A-E-L-D-L- p.H-S-G-I-V-Q-F-E-E-N-V-G-L-P-G-G-F-E-G-E-E-D-E-X-D-E-E-A-D-X-D-Q-E-I-A-X-V-T-

A-V-G-R-D-E-L-W-Y-F-L-A-E-I-A-L-R-R-L-L-N-R-V-S-Q-L-I-Y-S-K-D-T-P-Y-S-K-G-P-S-

M-A-S-T-T-S-L-E-P-I-V-A-E-L-D-F-Q-L-T-Q-W-Y-E (SEQ ID NO: 237). [0123] Clr-1 polypeptides of the present disclosure include, without limitation, the polypeptide sequences ofNCU07705 (SEQ ID NO: 1), XP_755084.1 (SEQ ID NO: 23), AN5808 (SEQ ID NO: 24), CAK44822.1 (SEQ ID NO: 25), BAE65369.1 (SEQ ID NO: 26), XP_001555641.1 (SEQ ID NO: 27), XP_001223845.1 (SEQ ID NO: 28), XP_385244.1 (SEQ ID NO: 29), EFQ33187.1 (SEQ ID NO: 30), EFX05743.1 (SEQ ID NO: 31), CBY01925.1 (SEQ ID NO: 32), XP_363808.2 (SEQ ID NO: 33), XP_003046557.1 (SEQ ID NO: 34), NCU00808 (SEQ ID NO: 35), XP_002561618.1 (SEQ ID NO: 36), XP 001793692.1 (SEQ ID NO: 37), XP_001910210.1 (SEQ ID NO: 38), XP_003302859.1 (SEQ ID NO: 39), XP__001941914.1 (SEQ ID NO: 40), XP_001586051.1 (SEQ ID NO: 41 ), XP 003349955.1 (SEQ ID NO: 42), SEQ ID NO: 43, XPJ)03009138.1 (SEQ ID NO: 44), XP_002147949.1 (SEQ ID NO: 45), XP_002481929.1 (SEQ ID NO: 46), EFY98315.1 (SEQ ID NO: 47), EGO59041.1 (SEQ ID NO: 48), XP 001267691.1 (SEQ ID NO: 15), XP_002378199.1 (SEQ ID NO: 16), CAK44822.1 (SEQ ID NO: 17), BAE65369.1 (SEQ ID NO: 18), XP_001209542.1 (SEQ ID NO: 19), EFY86844.1 (SEQ ID NO: 20), EGP86518.1 (SEQ ID NO: 21), XP_001260268.1 (SEQ ID NO: 22), and Trichoderma reesei clr-1 (SEQ ID NO: 182).

[0124] Clr-1 polypeptides of the present disclosure also include polypeptides that are homologs of clr-1 proteins identified herein. In some aspects, the present disclosure relates to polypeptides that are homologs of N. crassa clr-1 , homologs of Aspergillus nidulans clrA, and/or homologs of Trichoderma reesei clr-1. Methods for identification of polypeptides that are homologs of a polypeptide of interest are well known to one of skill in the art, as described herein.

[0125] Clr-1 polypeptides of the present disclosure further include polypeptides containing an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1 , SEQ ID NO: 24, or SEQ ID NO: 182. Polypeptides of the disclosure also include polypeptides having at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive amino acids of SEQ ID NO: 1 , SEQ ID NO: 24, or SEQ ID NO: 182.

[0126] A clr-1 polypeptide of the present disclosure includes, without limitation, clr-1 of Neurospora crassa (N. crassa), which has the gene name NCU07705 (SEQ ID NO: 1). The zinc(2)-cysteine(6) domain ofN. crassa clr-1 corresponds to about amino acids 134-166 of SEQ ID NO: 1. The conserved central domain of N. crassa clr-1 corresponds to about amino acids 313-549 of SEQ ID NO: 1. The zinc(2)-cysteine(6) domain and conserved central domain of other clr-1 polypeptides may be determined by aligning a clr-1 sequence of interest to the amino acid sequence of N. crassa clr-1 , and identifying the amino acids in a sequence of interest which align with amino acids 134-166 and 313-549 of SEQ ID NO: 1. Another clr-1 polypeptide of the present disclosure includes, without limitation, clrA of Aspergillus nidulans, which has the gene name AN5808 (SEQ ID NO: 24). A further clr-1 polypeptide of the present disclosure includes, without limitation, clr-1 of Trichoderma reesei (SEQ ID NO: 182).

Clr-1 Sequence Motifs

[0127] The amino acid sequences of N. crassa clr-1 and 22 clr-1 homologs were aligned with the MAFFT alignment algorithm, (CBRC mafft website) and alignments were manually inspected for regions of conservation outside of known conserved domains in likely orthologs, as determined by phylogenetic analysis. The analysis identified five conserved sequence motifs. The first conserved sequence is: A-G-D-[KR]-[LM]-I-[LI]-[ED]-[RKQH]-L-N-R-I-E-[SNG]-L- L (SEQ ID NO: 188). The second conserved sequence is: H-[HR]-[ADE]-G-H-[MLI]-P-Y-[IL]- [WF]-Q-G-A-L-S-[MI]-[VMI] (SEQ ID : 189). The third conserved sequence is: [NP]-[PS]- [LKTS]-K-[RK]-[RK]-[NSP]-[TSN]-[EDST]-X-X-[VIAT]-[DE]-Y-P (SEQ ID NO: 190), where X can be any amino acid residue. The fourth conserved sequence is: G-[GTSVN]-[FLI]-G-[TS]- W-[SNVAT]-[ANS]-[QTP]-[PA]-[TS] (SEQ ID NO: 191 ). The fifth conserved sequence is: R- [NH]-[LM]-[ST]-[QP]-[STP]-[SP]-[DE] (SEQ ID NO: 192). As an example of how to such motifs, the following motif, R-[NH]-[LM]-[ST]-[QP]-[STP]-[SP]-[DE] (SEQ ID NO: 192), is translated as: Arg-[Asn or His]-[Leu or Met]-[Ser or Thr]-[Gln or Pro]-[Ser, Thr, or Pro]-[Ser or Pro]-[Asp, or Glu] (SEQ ID NO: 192). These conserved motifs can be used to identify further clr-1 transcription factors.

[0128] Accordingly, in certain embodiments, clr-1 transcription factor proteins of the present disclosure contain a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequence selected from SEQ ID NOs: 188, 189, 190, 191, and 192.

Clr-2

[0129] In some aspects, the present disclosure relates to clr-2 polypeptides. Clr-2 polypeptides function as transcription factors that regulate the transcription of various genes in a fungal cell in response to the exposure of the cell to cellulose. In some aspects, the expression of a gene is increased in response to clr-2 expression. In some aspects, the expression of a gene is decreased in response to clr-2 expression.

[0130] Clr-2 is a member of the fungal specific zinc binuclear cluster superfamily, which is large, diverse superfamily of fungal-specific transcriptional regulators. Examples of transcription factors in this superfamily include gal-4, ace-1 , and xlnR (xyr-1) (Strieker AR, et al., App. Micro. Biotech., 78: 21 1 -220 (2008)). Clr-1 is also a member of this superfamily.

[0131] Members of this polypeptide superfamily typically contain two conserved domains: A) a zinc(2)-cysteine(6) binuclear cluster domain, which coordinates binding of the polypeptide to the DNA, and B) a central domain, which roughly corresponds to what is known as the "middle homology region" (Campbell RN, Biochemical J., 414: 177-187, (2008)), a conserved domain in zinc finger transcription factors. In clr-2, the conserved central domain has the fungal-specific transcription factor domain PFAM04082.

[0132] In certain embodiments, clr-2 polypeptides of the present disclosure have a zinc(2)- cysteine(6) binuclear cluster domain having the following conserved sequence: C-A-E-C-R-R-R- K-I-R-C-D-G-E-Q-PC-G-Q-C-X-W-Y-X-K-P-K-R-C-F-Y-R-V-X-P-S-R-K (SEQ ID NO: 236); and a PFAM04082 transcription factor domain having the following conserved sequence: 1-D-A- Y.F-K-R-V-H-X-F-X-P-M-L-D-E-X-T-F-R-A-T-Y-L-E-G-Q-R-K-D-A-P-W-L-A-L-L-N-M-V- F-A-L-G-S-I-A-A-M-K-S-D-D-Y-N-H-X-X-Y-Y-N-R-A-M-E-H-L-X-L-D-S-F-G-S-S-H-X-E- T-V-Q-A-L-A-L-M-G-G-Y-Y-L-H-Y-I-N-R-P-N-X-A-N-A-L-M-G-A-A-L-R-M-A-S-A-L-G-L- H-R-E-S-L-A-Q-X-X-A-S-S-Q-K-G-V-N-X-S-D-X-A-S-A-E-T-R-R-R-T-W-W-S-L-F-C-L-D- T-W-A-T-T-T-L-G-R-P-S-X-G-R-W-G (SEQ ID NO: 238).

[0133] Clr-2 polypeptides of the present disclosure include the polypeptide sequences of NCU08042 (SEQ ID NO: 4), CAE85541.1 (SEQ ID NO: 69), XP_003347695.1 (SEQ ID NO: 70), XP 001910304.1 (SEQ ID NO: 71), XP 001223809.1 (SEQ ID NO: 72), EFQ33148.1 (SEQ ID NO: 73), XPJ63907.1 (SEQ ID NO: 74), XPJ)03006605.1 (SEQ ID NO: 75), XP_003039508.1 (SEQ ID NO: 76), XP_001558061 .1 (SEQ ID NO: 77), XP_003299229.1 (SEQ ID NO: 78), CBX99480.1 (SEQ ID NO: 79), XP_001395273.2 (SEQ ID NO: 80), XP 384856.1 (SEQ ID NO: 81 ), XP 003191005.1 (SEQ ID NO: 82), XP_002568399.1 (SEQ ID NO: 83), EDP48079.1 (SEQ ID NO: 84), AN3369 (SEQ ID NO: 85), XP_ 003065241.1 (SEQ ID NO: 86), XP_001240945.1 (SEQ ID NO: 87), XP_002542864.1 (SEQ ID NO: 88), XP_002480618.1 (SEQ ID NO: 89), XP_001940688.1 (SEQ ID NO: 90), XP 002151678.1 (SEQ ID NO: 91), EFY98873.1 (SEQ ID NO: 92), XP_001590666.1 (SEQ ID NO: 93), EGR49862 (SEQ ID NO: 94), XP_961763.2 (SEQ ID NO: 95), EG059545.1 (SEQ ID NO: 96), SEQ ID NO: 97, CAK48469.1 (SEQ ID NO: 49), EFW 15774.1 (SEQ ID NO: 50),

XP_003040361.1 (SEQ ID NO: 51), XP_002561020.1 (SEQ ID NO: 52), XP_003009097.1 (SEQ ID NO: 53), XP_003001732.1 (SEQ ID NO: 54), XP_001272415.1 (SEQ ID NO: 55), XP_001268264.1 (SEQ ID NO: 56), XP_002384489.1 (SEQ ID NO: 57), XP_001217271.1 (SEQ ID NO: 58), XP_001214698.1 (SEQ ID NO: 59), XP_001218515.1 (SEQ ID NO: 60), EGP89821.1 (SEQ ID NO: 61), XP_001262768.1 (SEQ ID NO: 62), XP 001258355.1 (SEQ ID NO: 63), EDP49780.1 (SEQ ID NO: 64), XP_746801.1 (SEQ ID NO: 65), XP_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), and EFQ30604.1 (SEQ ID NO: 68).

[0134] Clr-2 polypeptides of the present disclosure also include polypeptides that are homologs of clr-2 proteins identified herein. In some aspects, the present disclosure relates to polypeptides that are homologs ofN. crassa clr-2 and/or homologs of Aspergillus nidulans clrB. Methods for identification of polypeptides that are homologs of a polypeptide of interest are well known to one of skill in the art, as described herein.

[0135] Clr-2 polypeptides of the present disclosure further include polypeptides containing an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 85.

Polypeptides of the disclosure also include polypeptides having at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive amino acids of SEQ ID NO: 4 or SEQ ID NO: 85.

[0136] A clr-2 polypeptide of the present disclosure includes, without limitation, clr-2 of N. crassa, which has the gene name NCU08042 (SEQ ID NO: 4). The zinc(2)-cysteine(6) domain of N. crassa clr-2 corresponds to about amino acids 48-86 of SEQ ID NO: 4. The conserved central domain of N. crassa clr-2 corresponds to about amino acids 271 -427 of SEQ ID NO: 4. The zinc(2)-cysteine(6) domain and conserved central domain of other clr-2 polypeptides may be determined by aligning a clr-2 sequence of interest to the sequence of N. crassa clr-2, and identifying the amino acids in a sequence of interest which align with amino acids 48-86 and 271-427 of SEQ ID NO: 4. Another clr-2 polypeptide of the present disclosure includes, without limitation, clrB of Aspergillus nidulans, which has the gene name AN3369 (SEQ ID NO: 85).

Clr-2 Sequence Motifs

[0137] The amino acid sequences of N. crassa clr-2 and 21 clr-2 homologs were aligned with the MAFFT alignment algorithm, (CBRC mafft website) and alignments were manually inspected for regions of conservation outside of known conserved domains in likely orthologs, as determined by phylogenetic analysis. The analysis identified five conserved sequence motifs. The analysis identified four conserved sequence motifs. The first conserved sequence is: [VL]- [ED]-[KAE]-L-S-[QTSN]-[STN]-[LVI]-[DE]-[DE]-[YC]-[RK]-[STV] (SEQ ID NO: 184). The second conserved sequence is: [MLI]-[STI]-G-W-N-A-V-W-[FLW]-[IVLCT]-[FY]-Q-[AS]-X- [ML]-[VI]-P-L-[ILV] (SEQ ID : 185), where X can be any amino acid residue. The third conserved sequence is: [ED]-X-L-[AV]-[AVI]-[STAL] (SEQ ID NO: 186), where X can be any amino acid residue. The fourth conserved sequence is: M-[FY]-[HIL]-T-F-[QE] (SEQ ID NO: 187). As an example of how to such motifs, the following motif, M-[FY]-[HIL]-T-F-[QE] (SEQ ID NO: 187), is translated as: Met-[Phe or Tyr]-[His, He, or Leu]-Thr-Phe-[Gln or Glu]. These conserved motifs can be used to identify further clr-2 transcription factors.

[0138] Accordingly, in certain embodiments, clr-2 transcription factor proteins of the present disclosure contain a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequence selected from SEQ ID NOs: 184, 185, 186, and 187.

Genes Under Regulatory Control of Clr-1 and Clr-2

[0139] Clr-1 and clr-2 function as transcription factors for genes involved in the detection and metabolic response of a cell to the presence of cellulose. In some aspects, clr-1 and clr-2 are involved in the regulation of genes encoding cellulases. In some aspects, clr-1 and clr-2 are involved in the regulation of genes encoding polysaccharide active enzymes. In some aspects, clr-1 and clr-2 are involved in the regulation of genes encoding transport proteins. In some aspects, clr-1 and clr-2 are involved in the regulation of genes encoding proteins involved in protein synthesis and/or secretion. In some aspects, clr-1 and clr-2 are involved in the regulation of genes encoding hemicellulases. Genes under the regulatory control of clr- 1 and/or clr-2are further described in Table 1A-1E. In some aspects, the expression of a gene under the control of clr-1 and/or clr-2 is increased in response to clr-1 and/or clr-2 expression. In some aspects, the expression of a gene under the control of clr-1 and/or clr-2 is decreased in response to clr-1 and/or clr-2 expression.

[0140] Advantageously, mis- expression of clr-2 in a filamentous fungal cell induces expression of one or more cellulase genes under non-inducing or starvation conditions, resulting in increased secretion of one or more cellulases from the cell. For example, the non-inducing or starvation conditions may include, without limitation, culturing the filamentous fungal cell in the absence of any easily usable carbon source, such as cellulose or cellobiose; and culturing the filamentous fungal cell in the presence of a preferred carbon source, such as sucrose.

[0141] As used herein, "mis-expression" of a gene refers to expression of a gene under conditions where the gene is not normally expressed, such as under non-inducing conditions. Mis-expression may include, without limitation, recombinant expression, constitutive expression, inducible expression, heterologous expression, and over-expression.

Polynucleotides of the Disclosure

[0142] The present disclosure further relates to polynucleotides that encode clr-1 and clr-2 polypeptides. Polynucleotides that encode a polypeptide are also referred to herein as "genes". Methods for determining the relationship between a polypeptide and a polynucleotide that encodes the polypeptide are well known to one of skill in the art. Similarly, methods of determining the polypeptide sequence encoded by a polynucleotide sequence are well known to one of skill in the art.

[0143] As used herein, the terms "polynucleotide", "nucleic acid sequence", "nucleic acid", and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D- ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog, and inter-nucleotide modifications. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC- IUB Commission of Biochemical Nomenclature.

Clr-1 [0144] The present disclosure relates to polynucleotides that encode a clr-1 polypeptide. In some aspects, the disclosure relates to polynucleotides that encode the polypeptides of

NCU07705 (SEQ ID NO: 1 ), XP_755084.1 (SEQ ID NO: 23), AN5808 (SEQ ID NO: 24), CAK44822.1 (SEQ ID NO: 25), BAE65369.1 (SEQ ID NO: 26), XP_001555641.1 (SEQ ID NO: 27), XP_001223845.1 (SEQ ID NO: 28), XP_385244.1 (SEQ ID NO: 29), EFQ33187.1 (SEQ ID NO: 30), EFX05743.1 (SEQ ID NO: 31), CBYO 1925.1 (SEQ ID NO: 32),

XP_363808.2 (SEQ ID NO: 33), XP_003046557.1 (SEQ ID NO: 34), NCU00808 (SEQ ID NO: 35), XP_002561618.1 (SEQ ID NO: 36), XP_001793692.1 (SEQ ID NO: 37), XP 001910210.1 (SEQ ID NO: 38), XP 003302859.1 (SEQ ID NO: 39), XP_001941914.1 (SEQ ID NO: 40), XP_001586051.1 (SEQ ID NO: 41), XP_003349955.1 (SEQ ID NO: 42), SEQ ID NO: 43, XP_003009138.1 (SEQ ID NO: 44), XP_002147949.1 (SEQ ID NO: 45), XP_002481929.1 (SEQ ID NO: 46), EFY98315.1 (SEQ ID NO: 47), EGO59041.1 (SEQ ID NO: 48),

XP_001267691.1 (SEQ ID NO: 15), XP_002378199.1 (SEQ ID NO: 16), CAK44822.1 (SEQ ID NO: 17), BAE65369.1 (SEQ ID NO: 18), XP_001209542.1 (SEQ ID NO: 19), EFY86844.1 (SEQ ID NO: 20), EGP86518.1 (SEQ ID NO: 21), XP_001260268.1 (SEQ ID NO: 22), and Trichoderma reesei clr-1 (SEQ ID NO: 182).

[0145] In some aspects, a polynucleotide of the disclosure is a polynucleotide that encodes the N. crassa clr-1 polypeptide. An example of a polynucleotide that encodes the N. crassa clr-1 polypeptide is SEQ ID NO: 2. In other aspects, a polynucleotide of the disclosure is a polynucleotide that encodes the Aspergillus nidulans clrA polypeptide. An example of a polynucleotide that encodes the Aspergillus nidulans clrA polypeptide is SEQ ID NO: 1 19. In further aspects, a polynucleotide of the disclosure is a polynucleotide that encodes the

Trichoderma reesei clr- 1 polypeptide. An example of a polynucleotide that encodes the Trichoderma reesei clr-1 polypeptide is SEQ ID NO: 183.

[0146] Polynucleotides of the disclosure also include polynucleotides having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%), at least 55%o, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183. Polynucleotides of the disclosure also include polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183. [0147] Polynucleotides of the disclosure further include fragments of polynucleotides that encode clr-1 polypeptides, polynucleotides that are complementary to polynucleotides that encode clr- 1 polypeptides, and fragments of polynucleotides that are complementary to polynucleotides that encode clr-1 polypeptides.

[0148] Polynucleotides of the disclosure also include polynucleotides that encode polypeptides containing an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1 , SEQ ID NO: 24, or SEQ ID NO: 182. Polynucleotides of the disclosure also include polynucleotides that encode polypeptides having at least 10, at least 12, at least 14, at least 16, at least 1 8, or at least 20 consecutive amino acids of SEQ ID NO: 1 , SEQ ID NO: 24, or SEQ ID NO: 182.

[0149] Polynucleotides of the disclosure that encode a clr-1 polypeptide also include polynucleotides having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 98-132. Polynucleotides of the disclosure also include polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 98-132.

Clr-2

[0150] The present disclosure relates to polynucleotides that encode a clr-2 polypeptide. In some aspects, the disclosure relates to polynucleotides that encode the polypeptides of

NCU08042 (SEQ ID NO: 4), CAE85541.1 (SEQ ID NO: 69), XP_003347695.1 (SEQ ID NO: 70), XPJ)01910304.1 (SEQ ID NO: 71 ), XPJ)01223809.1 (SEQ ID NO: 72), EFQ33148.1 (SEQ ID NO: 73), XP_363907.1 (SEQ ID NO: 74), XP_003006605.1 (SEQ ID NO: 75), XP_003039508.1 (SEQ ID NO: 76), XP_001558061.1 (SEQ ID NO: 77), XP_003299229.1 (SEQ ID NO: 78), CBX99480.1 (SEQ ID NO: 79), XP_001395273.2 (SEQ ID NO: 80), XPJ84856.1 (SEQ ID NO: 81), XPJ)03191005.1 (SEQ ID NO: 82), XPJ⁾02568399.1 (SEQ ID NO: 83), EDP48079.1 (SEQ ID NO: 84), AN3369 (SEQ ID NO: 85), XP_003065241.1 (SEQ ID NO: 86), XP_001240945.1 (SEQ ID NO: 87), XPJ)02542864.1 (SEQ ID NO: 88), XP_002480618.1 (SEQ ID NO: 89), XP _001940688.1 (SEQ ID NO: 90), XP_002151678.1 (SEQ ID NO: 91), EFY98873.1 (SEQ ID NO: 92), XPJX) 1590666.1 (SEQ ID NO: 93), EGR49862 (SEQ ID NO: 94), XP_961763.2 (SEQ ID NO: 95), EG059545.1 (SEQ ID NO: 96), SEQ ID NO: 97, CAK48469.1 (SEQ ID NO: 49), EFW15774.1 (SEQ ID NO: 50),

XP_003040361.1 (SEQ ID NO: 5 1 ), XP _002561020.1 (SEQ ID NO: 52), XP 003009097.1 (SEQ ID NO: 53), XP_003001732.1 (SEQ ID NO: 54), XPJX) 1272415.1 (SEQ ID NO: 55), XP 001268264.1 (SEQ ID NO: 56), XP_002384489.1 (SEQ ID NO: 57), XP_001217271.1 (SEQ ID NO: 58), XPJX) 1214698.1 (SEQ ID NO: 59), XP_001218515.1 (SEQ ID NO: 60), EGP89821.1 (SEQ ID NO: 61), XPJX) 1262768.1 (SEQ ID NO: 62), XP 001258355.1 (SEQ ID NO: 63), EDP49780.1 (SEQ ID NO: 64), XP_74680 U (SEQ ID NO: 65), XP_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), and EFQ30604.1 (SEQ ID NO: 68).

[0151] In some aspects, a polynucleotide of the disclosure is a polynucleotide that encodes the N. crassa clr-2 polypeptide. An example of polynucleotide that encodes the N. crassa clr-2 polypeptide is SEQ ID NO: 5. In other aspects, a polynucleotide of the disclosure is a polynucleotide that encodes the Aspergillus nidulans clrB polypeptide. An example of a polynucleotide that encodes the Aspergillus nidulans clrB polypeptide is SEQ ID NO: 165.

[0152] Polynucleotides of the disclosure also include polynucleotides having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 165. Polynucleotides of the disclosure also include polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of SEQ ID NO: 5 or SEQ ID NO: 165.

[0153] Polynucleotides of the disclosure further include fragments of polynucleotides that encode clr-2 polypeptides, polynucleotides that are complementary to polynucleotides that encode clr-2 polypeptides, and fragments of polynucleotides that are complementary to polynucleotides that encode clr-2 polypeptides.

[0154] Polynucleotides of the disclosure also include polynucleotides that encode polypeptides containing an amino acid sequence having at least 10%), at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%o, at least 75%, at least 80%, at least 85%o, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 85. Polynucleotides of the disclosure also include polynucleotides that encode polypeptides having at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive amino acids of SEQ ID NO: 4 or SEQ ID NO: 85.

[0155] Polynucleotides of the disclosure that encode a clr-2 polypeptide also include polynucleotides having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%), or 100% identity to any of the sequences of SEQ ID NOs: 133-181. Polynucleotides of the disclosure also include polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 133-181.

Sequence Homologs

[0156] As used herein, "homologs" are polypeptide or polynucleotide sequences that share a significant degree of sequence identity or similarity. Sequences that are homologs are referred to as being "homologous" to each other. Homologs include sequences that are orthologs or paralogs.

[0157] As used herein, "orthologs" are evolutionarily related polypeptide or polynucleotide sequences in different species that have similar sequences and functions, and that develop through a speciation event. Sequences that are orthologs are referred to as being "orthologous" to each other.

[0158] As used herein, "paralogs" are evolutionarily related polypeptide or polynucleotide sequences in the same organism that have similar sequences and functions, and that develop through a gene duplication event. Sequences that are paralogs are referred to as being

"paralogous" to each other. Methods of Identification of Homologous Sequences / Sequence Identity and Similarity

[0159] Several different methods are known to those of skill in the art for identifying homologous sequences, including phylogenetic methods, sequence similarity analysis, and hybridization methods.

Phylogenetic methods

[0160] Phylogenetic trees may be created for a gene family by using a program such as CLUSTAL (Thompson et al. Nucleic Acids Res. 22: 4673-4680 (1994); Higgins et al. Methods Enzymol 266: 383-402 (1996)) or MEGA (Tamura et al. Mol. Biol. & Evo. 24: 1596- 1599 (2007)). Once an initial tree for genes from one species is created, potential orthologous sequences can be placed in the phylogenetic tree and their relationships to genes from the species of interest can be determined. Evolutionary relationships may also be inferred using the Neighbor- Joining method (Saitou and Nei, Mol. Biol. & Evo. 4:406-425 (1987)). Homologous sequences may also be identified by a reciprocal BLAST strategy. Evolutionary distances may be computed using the Poisson correction method (Zuckerkandl and Pauling, pp. 97-166 in Evolving Genes and Proteins, edited by V. Bryson and H.J. Vogel. Academic Press, New York (1965)).

[0161] In addition, evolutionary information may be used to predict gene function.

Functional predictions of genes can be greatly improved by focusing on how genes became similar in sequence (i.e. by evolutionary processes) rather than on the sequence similarity itself (Eisen, Genome Res. 8: 163- 167 (1998)). Many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, Genome Res. 8: 163-167 (1998)). By using a phylogenetic analysis, one skilled in the art would recognize that the ability to deduce similar functions conferred by closely-related polypeptides is predictable.

[0162] When a group of related sequences are analyzed using a phylogenetic program such as CLUSTAL, closely related sequences typically cluster together or in the same clade (a group of similar genes). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle, J. Mol. Evol. 25: 351 -360 (1987)). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount, Bioinformatics: Sequence and Genome Analysis Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 543 (2001)).

[0163] To find sequences that are homologous to a reference sequence, BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the disclosure. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI- BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs (e.g. , BLASTN for nucleotide sequences, BLASTX for proteins) can be used.

Sequence Alignment / Sequence Similarity and Identity Analysis

[0164] Methods for the alignment of sequences and for the analysis of similarity and identity of polypeptide and polynucleotide sequences are well known in the art.

[0165] As used herein "sequence identity" refers to the percentage of residues that are identical in the same positions in the sequences being analyzed. As used herein "sequence similarity" refers to the percentage of residues that have similar biophysical / biochemical characteristics in the same positions (e.g. charge, size, hydrophobicity) in the sequences being analyzed.

[0166] Methods of alignment of sequences for comparison are well-known in the art, including manual alignment and computer assisted sequence alignment and analysis. This latter approach is a preferred approach in the present disclosure, due to the increased throughput afforded by computer assisted methods. As noted below, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.

[0167] The determination of percent sequence identity and/or similarity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS 4: 1 1-17 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); the search-for- similarity-method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444-2448 (1988); the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993).

[0168] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity and/or similarity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from

Intelligenetics, Mountain View, Calif.); the AlignX program, version 10.3.0 (Invitrogen, Carlsbad, CA) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. Gene 73:237-244 (1988); Higgins et al. CABIOS 5: 151-153 (1989); Corpet et al., Nucleic Acids Res. 16: 10881 -90

(1988) ; Huang et al. CABIOS 8: 1 55-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-331 (1994). The BLAST programs of Altschul et al. J. Mol. Biol. 215:403-410 (1990) are based on the algorithm of Karlin and Altschul (1990) supra.

Hybridization methods

[0169] Polynucleotides homologous to a reference sequence can be identified by

hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical- chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in references cited below (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. ("Sambrook")

(1989) ; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152 Academic Press, Inc., San Diego, Calif. ("Berger and Kimmel") (1987); and Anderson and Young, "Quantitative Filter Hybridisation." In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, TRL Press, 73-1 1 1 (1985)). [0170] Encompassed by the disclosure are polynucleotide sequences that are capable of hybridizing to the disclosed polynucleotide sequences, including any polynucleotide within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger, Methods Enzymol. 152: 399-407 (1987); and Kimmel, Methods Enzymo. 152: 507-51 1 , (1987)). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known polynucleotide hybridization methods.

[0171] With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) (supra); Berger and Kimmel (1987) pp. 467-469 (supra); and Anderson and Young (1985)(supra).

[0172] Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young (1985)(supra)). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecyl sulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.

[0173] Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency. As a general guidelines high stringency is typically performed at T_m-5°C to T_m-20°C, moderate stringency at T_m-20°C to T_m-35°C and low stringency at T_m-35°C to T_m-50° C for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50°C below T_m), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T_m-25°C for DNA-DNA duplex and T_m- 15°C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.

[0174] High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH.

[0175] Hybridization and wash conditions that may be used to bind and remove

polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example: 6X SSC and 1 % SDS at 65°C; 50% formamide, 4X SSC at 42°C; 0.5X SSC to 2.0 X SSC, 0.1 % SDS at 50°C to 65°C; or 0.1 X SSC to 2X SSC, 0.1% SDS at 50°C - 65°C; with a first wash step of, for example, 10 minutes at about 42°C with about 20% (v/v) formamide in 0.1 X SSC, and with, for example, a subsequent wash step with 0.2 X SSC and 0.1% SDS at 65°C for 10, 20 or 30 minutes.

[0176] For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50° C. An example of a low stringency wash step employs a solution and conditions of at least 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1 % SDS over 30 min. Greater stringency may be obtained at 42°C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. 20010010913).

[0177] If desired, one may employ wash steps of even greater stringency, including conditions of 65°C -68°C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS, or about 0.2X SSC, 0.1 % SDS at 65° C and washing twice, each wash step of 10, 20 or 30 min in duration, or about 0.1 X SSC, 0.1% SDS at 65° C and washing twice for 10, 20 or 30 min. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3°C to about 5°C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6°C to about 9°C. [0178] Polynucleotide probes may be prepared with any suitable label, including a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization probes for detecting related polynucleotide sequences may be produced, for example, by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Host Cells of the Disclosure

[0179] The present disclosure further relates to host cells that contain a recombinant nucleic acid encoding a clr-1 polypeptide, clr-2 polypeptide, or clr-1 and clr-2 polypeptides.

[0180] "Host cell" and "host microorganism" are used interchangeably herein to refer to a living biological cell that can be transformed via insertion of recombinant DNA or RNA. Such recombinant DNA or RNA can be in an expression vector.

[0181] Any prokaryotic or eukaryotic host cell may be used in the present disclosure so long as it remains viable after being transformed with a sequence of nucleic acids. Preferably, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., transporters), or the resulting intermediates. Suitable eukaryotic cells include, but are not limited to, fungal, plant, insect or mammalian cells.

[0182] In some aspects, the host is a fungal strain. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi.

[0183] In some aspects, the host cell is fungus of the Ascomycota phylum. In some aspects, the host cell is of the genus Melarhizi m, Gibberella, Nectria, Magnaporthe, Neurospora, Sordaria, Chaetomium, Podospora, Verticillium, Glomerella, Grosmannia, Sclerotinia,

Botryotinia, Aspergillus, Aspergillus, Penicillium, Leptosphaeria, Phaeosphaeria, Pyrenophora, Penicillium, Talaromyces, Trichoderma, Uncinocarpus, Coccidioidesi, Saccharomyces, Schizosaccharomyces, Sporotrichum (Myceliophthora) , Thielevia, Acremonium, Yar owia, Hansenula, Kluyveromyces, Pichia, Mycosphaerella, Neosartorya, Thermomyces (Humicola, Monotospora, Sepedonium), or Chrysosporium.

[0184] In other aspects, the host cell is of the species Neurospora crassa, Metarhizium anisopliae, Metarhizium. acridum, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuc^iana, Aspergillus clavatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora Iritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila), Thielavia ierres/ra-thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum) , Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

[0185] The host cells of the present disclosure may be genetically modified in that recombinant nucleic acids have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing one or more nucleic acid constructs encoding one or more proteins for different functions.

[0186] "Recombinant nucleic acid" or "heterologous nucleic acid" or "recombinant polynucleotide", "recombinant nucleotide" or "recombinant DNA" as used herein refers to a polymer of nucleic acids where at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e. , not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids contains two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present disclosure describes the introduction of an expression vector into a host cell, where the expression vector contains a nucleic acid sequence coding for a protein that is not normally found in a host cell or contains a nucleic acid coding for a protein that is normally found in a cell but is under the control of different regulatory sequences. With reference to the host cell's genome, then, the nucleic acid sequence that codes for the protein is recombinant. As used herein, the term "recombinant polypeptide" refers to a polypeptide generated from a "recombinant nucleic acid" or "heterologous nucleic acid" or "recombinant polynucleotide", "recombinant nucleotide" or "recombinant DNA" as described above.

[0187] In some aspects, the host cell naturally produces any of the proteins encoded by the polynucleotides of the disclosure. The genes encoding the desired proteins may be heterologous to the host cell or these genes may be endogenous to the host cell but are operatively linked to heterologous promoters and/or control regions that result in the higher expression of the gene(s) in the host cell.

Host cell components

[0188] In some aspects, host cells of the disclosure contain a recombinant nucleic acid encoding a clr-1 polypeptide and/or a recombinant nucleic acid encoding a clr-2 polypeptide. In certain embodiments, the recombinant recombinant nucleic acid encoding a clr-1 polypeptide and/or recombinant nucleic acid encoding a clr-2 polypeptide is mis-expressed in the host cell (e.g., constitutively expressed, inducibly expressed, etc.). In other embodiments, a host cell that contains a recombinant nucleic acid encoding a clr-1 polypeptide and/or a recombinant nucleic acid encoding a clr-2 polypeptide contains a greater amount of clr-1 polypeptide and/or clr-2 polypeptide than a corresponding host cell that does not contain a recombinant nucleic acid encoding a clr-1 polypeptide and/or a recombinant nucleic acid encoding a clr-2 polypeptide. When a protein or nucleic acid is produced or maintained in a host cell at an amount greater than normal, the protein or nucleic acid is "overexpressed". In some aspects, host cells of the disclosure overexpress clr-1 and/or clr-2. The present disclosure further is directed to cells that are modified and that have a greater level of clr- 1 and/or clr-2 polypeptide than a corresponding cell that is not modified.

[0189] In some aspects, host cell of the disclosure contain a recombinant nucleic acid encoding a clr-2 transcription factor protein that contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187. In certain embodiments, the host cell may further contain at least one additional recombinant nucleic acid encoding a clr-1 transcription factor protein that contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. [0190] In other aspects, host cell of the disclosure contain a recombinant nucleic acid encoding a clr-1 transcription factor protein that contains a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, at least four, or at least five polypeptide sequences selected from SEQ ID NOs: 188, 189, 190, 191 , and 192. In certain embodiments, the host cell may further contain at least one additional recombinant nucleic acid encoding a cIr-2 transcription factor protein that contains a zinc(2)- cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one, at least two, at least three, or at least four polypeptide sequences selected from SEQ ID NOs: 184, 185, 186, and 187.

[0191] In some aspects, host cells of the disclosure contain a recombinant nucleic acid encoding a clr-1 polypeptide. In some aspects, host cells contain a recombinant nucleic acid encoding a clr-1 polypeptide having the amino acid sequence of any of: NCU07705 (SEQ ID NO: 1 ), XPJ755084.1 (SEQ ID NO: 23), AN5808 (SEQ ID NO: 24), CAK44822.1 (SEQ ID NO: 25), BAE65369.1 (SEQ ID NO: 26), XP 00155564 U (SEQ ID NO: 27), XP_001223845.1 (SEQ ID NO: 28), XP_385244.1 (SEQ ID NO: 29), EFQ33187.1 (SEQ ID NO: 30),

EFX05743.1 (SEQ ID NO: 31), CBY01925.1 (SEQ ID NO: 32), XPJ63808.2 (SEQ ID NO: 33), XPJ)03046557.1 (SEQ ID NO: 34), NCU00808 (SEQ ID NO: 35), XP_002561618.1 (SEQ ID NO: 36), XPJXH 793692.1 (SEQ ID NO: 37), XP_001910210.1 (SEQ ID NO: 38),

XP_003302859.1 (SEQ ID NO: 39), XP 001941914.1 (SEQ ID NO: 40), XP 001586051.1 (SEQ ID NO: 41), XPJ)03349955.1 (SEQ ID NO: 42), SEQ ID NO: 43, XP_003009138.1 (SEQ ID NO: 44), XP_002147949.1 (SEQ ID NO: 45), XP_ 002481929.1 (SEQ ID NO: 46),

EFY98315.1 (SEQ ID NO: 47), EGO59041.1 (SEQ ID NO: 48), XP 001267691.1 (SEQ ID NO: 15), XPJ)02378199.1 (SEQ ID NO: 16), CAK44822.1 (SEQ ID NO: 17), BAE65369.1 (SEQ ID NO: 18), XP_001209542.1 (SEQ ID NO: 19), EFY86844.1 (SEQ ID NO: 20), EGP86518.1 (SEQ ID NO: 21), XP 001260268.1 (SEQ ID NO: 22), or Trichoderma reesei clr-1 (SEQ ID NO: 182).

[0192] In some aspects, host cells contain a recombinant nucleic acid having the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 1 19, SEQ ID NO: 183, or any of SEQ ID NOs: 98- 132.

[0193] In some aspects, host cells of the disclosure contain a recombinant nucleic acid encoding a clr-2 polypeptide. In some aspects, host cells contain a recombinant nucleic acid encoding a clr-2 polypeptide having the amino acid sequence of any of: NCU08042 (SEQ ID NO: 4), CAE85541.1 (SEQ ID NO: 69), XP_003347695.1 (SEQ ID NO: 70), XP_001910304.1 (SEQ ID NO: 71), XP_001223809.1 (SEQ ID NO: 72), EFQ33148.1 (SEQ ID NO: 73), XPJ63907.1 (SEQ ID NO: 74), XP_003006605.1 (SEQ ID NO: 75), XP_003039508.1 (SEQ ID NO: 76), XP_001558061.1 (SEQ ID NO: 77), XP_003299229.1 (SEQ ID NO: 78),

CBX99480.1 (SEQ ID NO: 79), XP_001395273.2 (SEQ ID NO: 80), XP_384856.1 (SEQ ID NO: 81), XP_003191005.1 (SEQ ID NO: 82), XP 002568399.1 (SEQ ID NO: 83), EDP48079.1 (SEQ ID NO: 84), AN3369 (SEQ ID NO: 85), XP_003065241 .1 (SEQ ID NO: 86),

XP_001240945.1 (SEQ ID NO: 87), XP_002542864.1 (SEQ ID NO: 88), XP_002480618.1 (SEQ ID NO: 89), XP_001940688.1 (SEQ ID NO: 90), XP_002151678.1 (SEQ ID NO: 91 ), EFY98873.1 (SEQ ID NO: 92), XP_001590666.1 (SEQ ID NO: 93), EGR49862 (SEQ ID NO: 94), XP 961763.2 (SEQ ID NO: 95), EG059545.1 (SEQ ID NO: 96), SEQ ID NO: 97, CAK48469.1 (SEQ ID NO: 49), EFW15774.1 (SEQ ID NO: 50), XP_003040361.1 (SEQ ID NO: 51), XP_002561020.1 (SEQ ID NO: 52), XP_003009097.1 (SEQ ID NO: 53),

XP_003001732.1 (SEQ ID NO: 54), XP 001272415.1 (SEQ ID NO: 55), XP_001268264.1 (SEQ ID NO: 56), XP_002384489.1 (SEQ ID NO: 57), XP_001217271.1 (SEQ ID NO: 58), XP_001214698.1 (SEQ ID NO: 59), XP 001218515.1 (SEQ ID NO: 60), EGP89821.1 (SEQ ID NO: 61 ), XP_001262768.1 (SEQ ID NO: 62), XP_001258355.1 (SEQ ID NO: 63), EDP49780.1 (SEQ ID NO: 64), XP 746801.1 (SEQ ID NO: 65), XP_7 1092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), or EFQ30604.1 (SEQ ID NO: 68).

[0194] In some aspects, host cells contain a recombinant nucleic acid having the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 165, or any of SEQ ID NOs: 133-181.

[0195] In some aspects, host cells of the current disclosure contain recombinant nucleic acids encoding a clr-1 polypeptide and a clr-2 polypeptide. In some aspects, host cells of the present disclosure contain recombinant nucleic acids encoding a clr-1 polypeptide having the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 24, or SEQ ID NO: 182, and a clr-2 polypeptide having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 85. In some aspects, host cells of the current disclosure contain recombinant nucleic acids having the nucleic acid sequences of SEQ ID NO: 2, SEQ ID NO: 1 19, or SEQ ID NO: 183, and SEQ ID NO: 5 or SEQ ID NO: 165.

[0196] Host cells of the disclosure may also be modified to reduce or inhibit expression of at least one gene involved in regulating protein secretion to increase secretion of proteins, such as cellulases. In some embodiments, the host cell is modified to reduce or inhibit expression of the catabolite repressor gene cre-1, or a homolog thereof. Techniques for modifying cells to reduce or inhibit expression of a gene are well known in the art and include, without limitation, those disclosed herein. Non-limiting examples include mutagenesis, RNAi, and antisense suppression.

[0197] Host cells of the disclosure may further contain one or more recombinant nucleic acid sequences encoding a hemicellulase. Hemicellulases include, without limitation, exoxylanases, endoxylanases, D-arabinofuranosidases,□ -glucuronidases, D-xylosidases, and acetyl xylan esterases.

[0198] Host cells of the disclosure may further contain one or more recombinant nucleic acid sequences that encode a polypeptide in a biochemical pathway related to the production of a biofuel. In some aspects, a host cell contains a recombinant nucleic acid sequence encoding a polypeptide in a biochemical pathway involved in the production of ethanol, n-propanol, n- butanol, iso-butanol, 3-methyl-l -butanol, 2-methyl-l-butanol, 3-methyl-l -pentanol, and/or octanol.

Methods of Producing and Culturing Host Cells of the Disclosure

[0199] Methods of producing and culturing host cells of the disclosure may include the introduction or transfer of expression vectors containing the recombinant nucleic acids of the disclosure into the host cell. Such methods for transferring expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming cells with an expression vector involves a calcium chloride treatment where the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Cells also may be transformed through the use of spheroplasts (Schweizer, M, Proc. Natl. Acad. Sci., 78: 5086-5090 (1981 ). Also, microinjection of the nucleic acid sequences provides the ability to transfect host cells. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.

[0200] In some cases, cells are prepared as protoplasts or spheroplasts prior to

transformation. Protoplasts or spheroplasts may be prepared, for example, by treating a cell having a cell wall with enzymes to degrade the cell wall. Fungal cells may be treated, for example, with chitinase. [0201] The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host, or a transposon may be used.

[0202] The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene the product of which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selection of bacterial cells may be based upon antimicrobial resistance that has been conferred by genes such as the amp, gpt, neo, and hyg genes.

[0203] Selectable markers for use in fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5 '-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Suitable markers for S. cerevisiae hosts are, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.

[0204] The vectors may contain an element(s) that permits integration of the vector into the host's genome or autonomous replication of the vector in the cell independent of the genome.

[0205] For integration into the host genome, the vector may rely on the gene's sequence or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host. The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, or 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host. Furthermore, the integrational elements may be non- encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.

[0206] For autonomous replication, the vector may further contain an origin of replication enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a sequence that enables a plasmid or vector to replicate in vivo.

[0207] The vector may further contain a promoter for regulation of expression of a recombinant nucleic acid of the disclosure in the vector. Promoters for the regulation of expression of a gene are well-known in the art, and include constitutive promoters, and inducible promoters. Promoters are described, for example, in Sambrook, et al. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, (2001). Promoter can be viral, bacterial, fungal, mammalian, or plant promoters. Additionally, promoters can be constitutive promoters, inducible promoters, environmentally regulated promoters, or developmentally regulated promoters. Examples of suitable promoters for regulating recombinant nucleic acid of the disclosure, such as clr-1 and clr-2, include, without limitation, the N. crassa ccg-1 constitutive promoter, which is responsive to the N. crassa circadian rhythm and nutrient conditions; the N. crassa gpd- 1 (glyceraldehyde 3-phosphate dehydrogenase- ! ) strong constitutive promoter; the N. crassa vvd (light) inducible promoter; the N. crassa qa-2 (quinic acid) inducible promoter; the Aspergillus nidulans gpdA promoter; the Aspergillus nidulans trpC constitutive promoter; the N. crassa tef- 1 (transcription elongation factor) highly constitutive promoter; and the N. crassa xlr-1 (XlnR homolog) promoter, which is used frequently in Aspergillus species.

[0208] More than one copy of a gene may be inserted into the host to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. [0209] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0210] The host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.

[0211] Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. Growth of a host cell in a medium may involve the process of fermentation. Methods of the disclosure may include culturing the host cell such that recombinant nucleic acids in the cell are expressed. Media, temperature ranges and other conditions suitable for growth are known in the art.

[0212] According to some aspects of the disclosure, the culture media contains a carbon source for the host cell. Such a "carbon source" generally refers to a substrate or compound suitable to be used as a source of carbon for cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides,

oligosaccharides, polysaccharides, a biomass polymer such as cellulose or hemicellulose, xylose, arabinose, disaccharides, such as sucrose, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof.

[0213] In addition to an appropriate carbon source, media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for the fermentation of various sugars and the production of hydrocarbons and hydrocarbon derivatives. Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism. As the host cell grows and/or multiplies, expression of the enzymes, transporters, or other proteins necessary for growth on various sugars or biomass polymers, sugar fermentation, or synthesis of hydrocarbons or hydrocarbon derivatives is affected. Cells with Reduced Expression of Clr- 1 , Clr-2, or Clr- 1 and Clr-2

[0214] The present disclosure also relates to cells that naturally produce clr- 1 and clr-2 polypeptides and cellulase enzymes ("cellulolytic cells"), which have a reduced level of expression of clr-1 , clr-2, or clr-1 and clr-2. Cells that naturally produce cellulase enzymes and that have a reduced level of expression of clr-1 , clr-2, or clr-1 and clr-2 may have reduced levels of expression or secretion of one or more cellulases. Without being bound by theory, cells that naturally produce cellulase enzymes which have a reduced level of expression of clr-1 , clr-2, or clr-1 and clr-2 may have reduced levels of expression or secretion of one or more cellulases due to reduced activity of clr-1, clr-2 or clr-1 and clr-2 as transcription factors promoting the transcription of cellulase genes. The level of expression of a gene may be assessed by measuring the level of mRNA encoded by the gene, and/or by measuring the level or activity of the polypeptide encoded by the gene.

[0215] Furthermore, provided herein are methods of preparing cells which have a reduced level of expression clr-1 , clr-2, or both clr-1 and clr-2. Reduction in gene expression may be achieved by any number of techniques well known in the art, including without limitation, mutagenesis, RNAi, and antisense suppression.

Mutagenesis

[0216] Mutagenesis approaches may be used to disrupt or "knockout" the expression of a target gene. In some aspects, the mutagenesis results in a partial deletion of the target gene. In other aspects, the mutagenesis results in a complete deletion of the target gene. Methods of mutagenizing microorganisms, such as cellulolytic cells, are well known in the art and include, without limitation random mutagenesis and site-directed mutagenesis. Examples of methods of random mutagenesis include, without limitation, chemical mutagenesis (e.g., using ethane methyl sulfonate), insertional mutagenesis, and irradiation.

[0217] One method for reducing or inhibiting the expression of a target gene is by genetically modifying the target gene and introducing it into the genome of a cellulolytic cell to replace the wild-type version of the gene by homologous recombination (for example, as described in U.S. Pat. No. 6,924, 146).

[0218] Another method for reducing or inhibiting the expression of a target gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens, or transposons (see Winkler et al., Methods Mol. Biol. 82: 129-136, 1989, and Martienssen Proc. Natl. Acad. Sci. 95:2021-2026, 1998). After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a target gene.

[0219] Other methods to disrupt a target gene include insertional mutagenesis (for example, as described in U.S. Pat. No. 5,792,633), and transposon mutagenesis (for example, as described in U.S. Pat. No. 6,207,384)

[0220] A further method to disrupt a target gene is by use of the cre-lox system (for example, as described in U.S. Pat. No. 4,959,317).

[0221] Another method to disrupt a target gene is by use of PCR mutagenesis (for example, as described in U.S. Pat. No. 7,501 ,275).

RNAi

[0222] Endogenous gene expression may also be reduced or inhibited by means of RNA interference (RNAi), which uses a double-stranded RNA having a sequence identical or similar to the sequence of the target gene. As used herein RNAi, includes the use of micro RNA, such as artificial miRNA to suppress expression of a gene.

[0223] RNAi is the phenomenon in which when a double-stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA.

[0224] Thus, in some aspects, reduction or inhibition of gene expression is achieved using RNAi techniques. For example, to achieve reduction or inhibition of the expression of a DNA encoding a protein using RNAi, a double-stranded RNA having the sequence of a DNA encoding the protein, or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a cellulolytic cell of interest. As used herein, RNAi and dsRNA both refer to gene-specific silencing that is induced by the introduction of a double-stranded RNA molecule, see e.g., U.S. Pat. Nos. 6,506,559 and 6,573,099, and includes reference to a molecule that has a region that is double-stranded, e.g., a short hairpin RNA molecule. The resulting cellulolytic cells may then be screened for a phenotype associated with the reduced expression of the target gene, e.g., reduced cellulase expression, and/or by monitoring steady-state RNA levels for transcripts of the target gene. Although the sequences used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the target gene sequence. See, e.g., U.S. Patent Application Publication No. 2004/0029283. The constructs encoding an RNA molecule with a stem-loop structure that is unrelated to the target gene and that is positioned distally to a sequence specific for the gene of interest may also be used to inhibit target gene expression. See, e.g., U.S. Patent Application Publication No. 2003/022121 1.

[0225] The RNAi nucleic acids may encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, or 500 nucleotides corresponding to the target sequence. In addition, in some aspects, these fragments are at least, e.g., 50, 100, 150, 200, or more nucleotides in length.

Interfering RNAs may be designed based on short duplexes (i.e., short regions of double- stranded sequences). Typically, the short duplex is at least about 15, 20, or 25-50 nucleotides in length (e.g., each complementary sequence of the double stranded RNA is 15-50 nucleotides in length), often about 20-30 nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, fragments for use in RNAi will correspond to regions of a target protein that do not occur in other proteins in the organism or that have little similarity to other transcripts in the organism, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases. Similarly, RNAi fragments may be selected for similarity or identity with a conserved sequence of a gene family of interest, such as those described herein, so that the RNAi targets multiple different gene transcripts containing the conserved sequence.

[0226] RNAi may be introduced into a cellulolytic cell as part of a larger DNA construct. Often, such constructs allow stable expression of the RNAi in cells after introduction, e.g., by integration of the construct into the host genome. Thus, expression vectors that continually express RNAi in cells transfected with the vectors may be employed for this disclosure. For example, vectors that express small hairpin or stem-loop structure RNAs, or precursors to microRNA, which get processed in vivo into small RNAi molecules capable of carrying out gene-specific silencing (Brummelkamp et al, Science 296:550-553, (2002); and Paddison, et al., Genes & Dev. 16:948-958, (2002)) can be used. Post-transcriptional gene silencing by double- stranded RNA is discussed in further detail by Hammond et al, Nature Rev Gen 2: 1 10-1 19, (2001); Fire et al., Nature 391 : 806-81 1 , (1998); and Timmons and Fire, Nature 395 : 854, (1998). [0227] Methods for selection and design of sequences that generate RNAi are well known in the art (e.g. U.S. Pat. Nos. 6,506,559; 6,51 1,824; and 6,489, 127).

[0228] In some aspects, RNAi sequences used herein correspond to a portion of SEQ ID NO: 2, SEQ ID NO: 1 19, SEQ ID NO: 183, SEQ ID NO: 5, or SEQ ID NO: 165. In some aspects, RNAi sequences used herein correspond to a portion of a nucleotide sequence having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 2, SEQ IDNO: 1 19, SEQ ID NO: 183, SEQ ID NO: 5, or SEQ ID NO: 165. In some aspects, RNAi sequences used herein correspond to a portion of a nucleotide sequence having at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of SEQ ID NO: 2, SEQ IDNO: 1 1 , SEQ ID NO: 183, SEQ ID NO: 5, or SEQ ID NO: 165.

[0229] One of skill in the art will recognize that using technology based on specific nucleic acid sequences, families of homologous genes can be suppressed with a single transcript. For instance, if an antisense transcript is designed to have a sequence that is conserved among a family of genes, then multiple members of a gene family can be suppressed. Conversely, if the goal is to only suppress one member of a homologous gene family, then the transcript should be targeted to sequences with the most variation between family members.

[0230] The term "target gene" or "target sequences", refers to a gene targeted for reduced expression.

Antisense and ribozyme suppression

[0231] A reduction or inhibition of gene expression in a cellulolytic cell of a target gene may also be obtained by introducing into cellulolytic cells antisense constructs based on a target gene nucleic acid sequence. For antisense suppression, a target sequence is arranged in reverse orientation relative to the promoter sequence in the expression vector. The introduced sequence need not be a full length cDNA or gene, and need not be identical to the target cDNA or a gene found in the cellulolytic cell to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native target sequence is used to achieve effective antisense suppression. In some aspects, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. In some aspects, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from an endogenous target gene.

Suppression of a target gene expression can also be achieved using a ribozyme. The production and use of ribozymes are disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508.

Cellulolytic cells having multiple target genes inhibited

[0232] Expression of at least two target genes may be reduced or inhibited in a cellulolytic cell as described herein. In some aspects, both clr-1 and clr-2 genes are inhibited. In cells where expression of both clr-1 and clr-2 are reduced or inhibited, the same technique (e.g. RNAi, mutagenesis, etc.) may be used to reduce the expression of both clr-1 and clr-2, or different techniques may be used to reduce the expression of each of clr-1 and clr-2.

[0233] In further aspects at least one additional gene involved in regulating protein secretion, such as cellulase secretion, may be reduced or inhibited in a cellulolytic cell as described herein. In some embodiments, the catabolite repressor gene cre-1 is reduced or inhibited in the cellulolytic cell. In cells where expression of cre-1 in combination with clr-1 and/or clr-2 is reduced or inhibited, the same technique (e.g., RNAi, mutagenesis, etc.) may be used to reduce expression of cre-1, and clr- 1 and/or clr-2. Alternatively, different techniques may be used to reduce the expression of each of cre-1 , and clr-1 and/or clr-2.

Expression of target gene inhibitors

[0234] Expression cassettes containing nucleic acids that encode target gene expression inhibitors, e.g., an antisense or siRNA, can be constructed using methods well known in the art. Constructs include regulatory elements, including promoters and other sequences for expression and selection of cells that express the construct. Typically, fungal and/or bacterial transformation vectors include one or more cloned coding sequences (genomic or cDNA) under the

transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal. [0235] In certain aspects, a cell which has a reduced level of expression clr-1, clr-2, or both clr-1 and clr-2 is fungus of the Ascomycota phylum. In some aspects, the cell which has a reduced level of expression clr-1, clr-2, or both clr-1 and clr-2 is of the genus Metarhizium, Gibberella, Nectria, Magnaporthe, Neurospora, Sordaria, Chaetomium, Podospora, Verticillium, Glomerella, Grosmannia, Sclerotinia, Botryotinia, Aspergillus, Aspergillus, Penicillium, Leptosphaeria, Phaeosphaeria, Pyrenophora, Penicillium, Talaromyces, Trichoderma,

Uncinocarpus, Coccidioidesi, Saccharomyces, Schizosaccharomyces, Sporotrichum

(Myceliophthora), Thielevia, Acremonium, Yarrowia, Hansenula, Kluyveromyces, Pichia, Mycosphaerella, Neosartorya, Thermomyces (Humicola, Monotospora, Sepedonium), or Chrysosporium.

[0236] In some aspects, the cell which has a reduced level of expression clr-1 , clr-2, or both clr-1 and clr-2 is of the species Neurospora crassa, Metarhizium anisopliae, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora, Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum thermophile (Myceliophthora thermophila), Thielavia terrestris- thermophilic, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense.

Applications

Methods of Increasing Cell Growth

[0237] Provided herein are methods for increasing the growth rate of a cell having one or more genes encoding cellulases. In one aspect, a method for increasing the growth rate of a cell having one or more genes encoding cellulases includes increasing the expression of clr-1 , clr-2, or clr-1 and clr-2 polypeptides in the cell. Cells having increased expression of clr-1 , clr-2, or clr-1 and clr-2 polypeptides in the cell may have an increased growth rate as compared with a corresponding cell not having increased expression of clr-1, clr-2, or clr-1 and clr-2 polypeptides in the cell. Alternatively, the growth rate of a cell having one or more genes encoding cellulases may be increased by mis-expressing recombinant nucleic acids encoding clr-1 , clr-2, or clr-1 and clr-2 polypeptides in the cell. To increase the growth rate of a cell having one or more genes encoding cellulases, a cell containing recombinant nucleic acid(s) encoding clr-1 , clr-2, or clr-1 and clr-2 polypeptides is incubated in media under conditions sufficient to support the expression of clr-1 , clr-2, or clr-1 and clr-2. In some aspects, to increase the growth rate of a cell having one or more genes encoding cellulases, a cell containing recombinant nucleic acid(s) encoding clr-1 , clr-2, or clr-1 and clr-2 polypeptides is incubated in media containing cellulose under conditions sufficient to support the expression of clr-1, clr-2, or clr-1 and clr-2. In other aspects, expression of at least one gene involved in regulating protein secretion, such as cellulase secretion, is reduced or inhibited in the cell. In some embodiments, expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0238] Methods for increasing the growth rate of a cell disclosed herein apply to all host cells disclosed herein.

Methods of Degrading a Cellulose-Containing Material

[0239] Provided herein are methods for degrading a cellulose-containing material. In one aspect, a method for degrading a cellulose-containing material includes the steps of: A) contacting a cellulose-containing material with a fungal host cell having at least one recombinant nucleic acid encoding clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins in media under conditions necessary to support the expression of the at least one recombinant nucleic acid; and B) incubating the fungal host cell and cellulose-containing material under conditions t sufficient for the fungal host cell to degrade the cellulose-containing material. In certain embodiments, the fungal host cell is incubated under conditions sufficient for the fungal host cell to express said clr-2 transcription factor protein.

[0240] In another aspect, a method for degrading cellulose-containing material includes the steps of: A) incubating a fungal host cell having at least one recombinant nucleic acid encoding clr-1, clr-2, or clr- 1 and clr-2 transcription factor proteins in media under conditions necessary to support the expression of the at least one recombinant nucleic acid; B) collecting one or more cellulases from the media and/or cell; and C) incubating the one or more cellulases from the 8 media and/or cell with a cellulose-containing material under conditions sufficient for the one or more cellulases to degrade the t cellulose-containing material. In certain embodiments, the fungal host cell is incubated under conditions sufficient for the fungal host cell to express said clr-2 transcription factor protein.

[0241] In some embodiments, the fungal host cell produces a greater amount of one or more cellulases than a corresponding fungal host cell lacking the at least one recombinant nucleic acid.

[0242] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as cellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre- 1 is reduced or inhibited in the cell.

[0243] As used herein, a "cellulose-containing material" is any material that contains cellulose, including biomass, such as biomass containing plant material. Biomass suitable for use with the currently disclosed methods include any cellulose-containing material, and includes, without limitation, Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, rye hulls, wheat hulls, sugarcane bagasse, copra meal, copra pellets, palm kernel meal, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, energy cane, waste paper, sawdust, forestry wastes, municipal solid waste, waste paper, crop residues, other grasses, and other woods.

[0244] As an initial processing step in the degradation of biomass, biomass may be subjected to one or more pre-processing steps. Pre-processing steps are known to those of skill in the art, and include physical and chemical processes. Pre-processing steps include, without limitation, ammonia fiber expansion (AFEX), steam explosion, treatment with high temperature, treatment with high pressure, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.

[0245] In further embodiments, the fungal host cell may also have one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of at least one biofuel. Accordingly, the fungal host cell may also be incubated with degraded cellulose-containing material under conditions sufficient for the fungal host cell to convert the cellulose-containing material to at least one biofuel. Alternatively, the degraded cellulose- containing material may be cultured with a fermentative microorganism under conditions sufficient to produce at least one fermentation product from the degraded cellulose-containing material. Suitable biofuels and/or fermentation products include, without limitation, ethanol, n- propanol, n-butanol, iso-butanol, 3-methyl-l -butanol, 2-methyl-l-butanol, 3 -methyl- 1 -pentanol, and octanol.

Methods for Reduction of the Viscosity of Pretreated Biomass Mixtures

[0246] Also provided herein are methods for reducing the viscosity of pretreated biomass mixtures, prior to the degradation of the pretreated biomass mixtures into monosaccharides and oligosaccharides.

[0247] Biomass that is used for as a feedstock, for example, in biofuel production generally contains high levels of lignin, which can block hydrolysis of the cellulosic component of the biomass. Typically, biomass is subjected to a pretreatment step to increase the accessibility of the cellulosic component to hydrolysis. However, pretreatment generally results in a biomass mixture that is highly viscous. The high viscosity of the pretreated biomass mixture can also interfere with effective hydrolysis of the pretreated biomass. Advantageously, the cells of the present disclosure having an increased expression of clr-1 , clr-2, or clr-1 and clr-2 of the present disclosure, or cellulases produced from the cells, can be used to reduce the viscosity of pretreated biomass mixtures prior to further degradation of the biomass.

[0248] Accordingly, certain aspects of the present disclosure relate to methods of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with any of the cells having an increased expression of clr-1 , clr-2, or clr-1 and clr-2 of the present disclosure, or with cellulases produced from the cells, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture.

[0249] In some aspects, the disclosed methods are carried out as part of a pretreatment process. The pretreatment process may include the additional step of adding any of the cells having an increased expression of clr-1 , clr-2, or clr-1 and clr-2, of the present disclosure, or cellulases produced from the cells, to pretreated biomass mixtures after a step of pretreating the biomass, and incubating the pretreated biomass with the cells having an increased expression of clr-1 , clr-2, or clr-1 and clr-2, or cellulases produced from the cells, under conditions sufficient to reduce the viscosity of the mixture. The cells having an increased expression of clr-1 , clr-2, or clr-1 and clr-2, or the cellulases produced from the cells may be added to the pretreated biomass mixture while the temperature of the mixture is high, or after the temperature of the mixture has decreased. In some aspects, the methods are carried out in the same vessel or container where the pretreatment was performed. In other aspects, the methods are carried out in a separate vessel or container where the pretreatment was performed.

[0250] In some aspects, the methods are carried out in the presence of high salt, such as solutions containing saturating concentrations of salts, solutions containing sodium chloride (NaCl) at a concentration of at least at or about 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, or 4 M sodium chloride, or potassium chloride (KC1), at a concentration at or about 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M 3.0 M or 3.2 M KC1 and/or ionic liquids, such as 1 ,3-dimethylimidazolium dimethyl phosphate ([DMIMJDMP) or [EMIM]OAc, or in the presence of one or more detergents, such as ionic detergents (e.g., SDS, CHAPS), sulfydryl reagents, such as in saturating ammonium sulfate or ammonium sulfate between at or about 0 and 1 M. In other aspects, the methods are carried out over a broad temperature range, such as between at or about 20°C and 50°C, 25°C and 55°C, 30°C and 60°C, or 60°C and 1 10°C. In some aspects, the methods may be performed over a broad pH range, for example, at a pH of between about 4.5 and 8.75, at a pH of greater than 7 or at a pH of 8.5, or at a pH of at least 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 83.0, or 8.5.

Methods of Converting Cellulose-Containing Materials to Fermentation Product

[0251] Further provided herein are methods for converting cellulose-containing materials to a fermentation production. In one aspect, a method for converting a cellulose-containing material into a fermentation product includes the steps of: A) contacting a cellulose-containing material with a cell having at least one recombinant nucleic acid encoding clr- 1, clr-2, or clr-1 and clr-2 transcription factor proteins under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the cell expressing the at least one recombinant nucleic acid encoding clr-1 , clr-2, or clr-1 and clr-2 transcription factor proteins under conditions sufficient for the fungal host cell to degrade the cellulose-containing material, in order to obtain sugars; and C) culturing the sugars with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0252] In another aspect, a method for converting a cellulose-containing material into a fermentation product includes the steps of: A) incubating a cell having recombinant nucleic acids encoding clr-1, clr-2, or clr-1 and clr-2 polypeptides in media under conditions necessary to support the expression of the recombinant nucleic acids; B) collecting cellulases from the media and/or cell; C) incubating cellulases from the media and/or cell with a cellulose-containing material under conditions that support cellulose degradation, in order to obtain sugars; and D) culturing the sugars with a fermentative microorganism under conditions sufficient to produce a fermentation product.

[0253] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as cellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0254] Sugars that may be obtained from the degradation of cellulose-containing materials include, without limitation, glucose, cellobiose, xylose, arabinose, galactose, glucuronic acid, and mannose.

[0255] Fermentation products that may be produced from sugars obtained from the degradation of cellulose-containing materials include, without limitation, ethanol, n-propanol, n- butanol, iso-butanol, 3-methyl-l -butanol, 2-methyl-l-butanol, 3 -methyl- 1 -pen tanol, and octanol.

[0256] Fermentative organisms include, without limitation, Saccharomyces spp.

Methods of Consolidated Bioprocessing

[0257] Further provided herein are methods for converting cellulose-containing materials to a fermentation production, by consolidated bioprocessing. Consolidated bioprocessing combines enzyme generation, biomass hydrolysis, and biofuel production into a single stage. In one aspect, a method for converting a cellulose-containing material into a fermentation product by consolidated bioprocessing includes the steps of: A) contacting a cellulose-containing material with a cell having at least one recombinant nucleic acids encoding clr-1 , clr-2, or clr-1 and clr-2 transcription factor proteins and one or more recombinant nucleic acids encoding a polypeptide involved in a biochemical pathway for the production of a biofuel under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the cell expressing the recombinant nucleic acids encoding clr-1 , clr-2, or clr-1 and clr-2

transcription factor proteins and one or more recombinant nucleic acids encoding a polypeptide involved in a biochemical pathway for the production of a biofuel under conditions sufficient for the cell to degrade the cellulose-containing material and ferment the degraded cellulose- containing material, thereby producing a fermentation product.

[0258] In another aspect, a method for converting a cellulose-containing material into a fermentation product by consolidated bioprocessing includes the steps of: A) contacting a cellulose-containing material with a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 transcription factor proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, under conditions that support cellulose degradation and fermentation, in order to produce a fermentation product. In some aspects, in methods of consolidated bioprocessing involving a non-naturally occurring fungal cell, where the cell naturally contains genes encoding clr-1 and clr-2 proteins, and where the cell contains modifications causing reduced expression of one or both of the clr-1 and clr-2 proteins, as compared to the expression of the clr-1 and clr-2 proteins in a corresponding fungal cell lacking said modifications, the non- naturally occurring fungal cell further contains one or more recombinant nucleic acids encoding a cellulase.

[0259] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as cellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0260] Fermentation products that may be produced from sugars obtained from the degradation of cellulose-containing materials include, without limitation, ethanol, n-propanol, n- butanol, iso-butanol, 3-methyl-l-butanol, 2-methyl-l -butanol, 3-methyl-l -pentanol, and octanol.

Methods of Increasing the Production of Celluloses

[0261] Provided herein are methods for increasing the production of cellulases from a cell having genes encoding one or more cellulases. In one aspect, a method for increasing the production of cellulases from a cell having genes encoding one or more cellulases includes increasing the expression of clr-1 , clr-2, or clr-1 and clr-2 transcription factor proteins in the cell. Cells having increased expression of clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins in the cell may have an increased production of cellulases as compared with a corresponding cell not having increased expression of clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins in the cell. To increase the production of cellulases from a cell having one or more genes encoding cellulases, a cell containing recombinant nucleic acid(s) encoding clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins is incubated in media under conditions sufficient to support the expression of clr-1 , clr-2, or clr-1 and clr-2. In some aspects, to increase the production of cellulases from a cell having one or more genes encoding cellulases, a cell containing recombinant nucleic acid(s) encoding clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins is incubated in media containing cellulose under conditions sufficient to support the expression of clr-1 , clr-2, or clr-1 and clr-2.

[0262] In other aspects, a method of increasing the production of one or more cellulases from a fungal cell includes providing a fungal host cell having at least one recombinant nucleic acid encoding clr-1 , clr-2, or clr-1 and clr-2 transcription factor proteins; and culturing the host cell under conditions sufficient to support the expression of the at least one recombinant nucleic acid, where the fungal host cell produces a greater amount of the one or more cellulases than a corresponding host cell lacking the at least one recombinant nucleic acid.

[0263] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as cellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0264] In other embodiments, the fungal host cell is cultured in the absence of cellulose.

[0265] Methods for increasing the growth rate of a cell disclosed herein apply to all host cells disclosed herein.

Methods of Producing Cellulases

[0266] Also provided herein are methods for producing cellulases from a cell having genes encoding one or more cellulases. In one aspect, a method for producing cellulases from a cell having genes encoding one or more cellulases includes the steps of: A) incubating a cell having at least one recombinant nucleic acid encoding clr-1, clr-2, or clr-1 and clr-2 transcription factor proteins in media under conditions necessary to support the expression of the recombinant nucleic acids, and B) collecting cellulases from the media and/or cell. In some aspects, the media used for incubating a cell contains cellulose. In some aspects, the media used for incubating a cell does not contain cellulose.

[0267] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as cellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0268] Cellulases that may be produced by the methods provided herein include any enzyme having cellulose-degrading activity, including endocellulases, exocellulases, beta-glucosidases, oxidative cellulases, and cellulose phosphorylases.

[0269] Cellulases can be collected from the media and/or cell by any method for protein purification and/or concentration, which are well known in the art. Proteins may be purified, without limitation, by ammonium sulfate fractionation and liquid chromatography, including ion-exchange, affinity, size-exclusion, and hydrophobic interaction chromatography. Proteins may be concentrated, without limitation, by ammonium sulfate fractionation, liquid

chromatography, including ion-exchange, affinity, and hydrophobic interaction chromatography, and centrifugal ultrafiltration. Cells may be disrupted to release cellular content by any method known in the art, including mechanical, chemical, or enzymatic disruption.

[0270] Methods for producing cellulases from a cell having genes encoding one or more cellulases disclosed herein apply to all host cells disclosed herein.

Methods of Producing Hemicellulases

[0271] Also provided herein are methods for producing hemicellulases from a cell having genes encoding one or more hemicellulases. In one aspect, a method for producing

hemicellulases from a cell having genes encoding one or more hemicellulases includes the steps of: A) incubating a cell having at least one recombinant nucleic acid encoding clr-1 , clr-2, or clr- 1 and clr-2 transcription factor proteins in media under conditions necessary to support the expression of the recombinant nucleic acids, and B) collecting hemicellulases from the media and/or cell. In some aspects, the media used for incubating a cell contains hemicellulose. In some aspects, the media used for incubating a cell does not contain hemicellulose.

[0272] In some embodiments, the method further includes reducing or inhibiting expression of at least one gene involved in regulating protein secretion, such as hemicellulase secretion. In certain preferred embodiments, the method further includes reducing or inhibiting expression of the catabolite repressor gene cre-1 is reduced or inhibited in the cell.

[0273] Hemicellulases that may be produced by the methods provided herein include any enzyme having hemicellulose-degrading activity, including, without limitation, exoxylanases, endoxylanases, Π-arabinofuranosidases,□ -glucuronidases, D-xylosidases, and acetyl xylan esterases.

[0274] Hemicellulases can be collected from the media and/or cell by any method for protein purification and/or concentration, which are well known in the art. Proteins may be purified, without limitation, by ammonium sulfate fractionation and liquid chromatography, including ion-exchange, affinity, size-exclusion, and hydrophobic interaction chromatography. Proteins may be concentrated, without limitation, by ammonium sulfate fractionation, liquid

[0275] Methods for producing hemicellulases from a cell having genes encoding one or more hemicellulases disclosed herein apply to all host cells disclosed herein.

Methods of Analyzing Cellular Response to Cellulose and/or Genes Involved in Cellulose Metabolism

[0276] In yet another aspect, provided herein are methods for analyzing a cellular response to cellulose. In one aspect, a method for analyzing a cellular response to cellulose involves the steps of: A) Obtaining a cell which naturally produces clr-1 and/or clr-2 that is modified to reduce expression of clr-1 and/or clr-2; B) contacting the cell which naturally produces clr-1 and/or clr-2 that is modified to reduce expression of clr-1 and/or clr-2 with a cellulose containing-material; and C) analyzing one or more components of the cell, such as a polypeptide or a nucleic acid, in response to the cellulose-containing material. In some aspects, a cell which naturally produces clr-1 and/or clr-2 that is modified to reduce expression of clr-1 and/or clr-2 further contains a recombinant nucleic acid, which encodes a polypeptide involved in cellulose metabolism. In some aspects, a polypeptide involved in cellulose metabolism is a cellulase. In some aspects, in a cell which naturally produces clr-1 and/or clr-2 that is modified to reduce expression of clr-1 and/or clr-2, and that contains a recombinant nucleic acid encoding a polypeptide involved in cellulose metabolism, the biological activity of the polypeptide involved in cellulose metabolism may be analyzed.

[0277] Cells may be modified to reduce the expression of clr-1 and/or clr-2 by any method disclosed herein for the reduction of expression of a gene

EXAMPLES

[0278] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

Example 1 : Induction of Cellulose Degrading Enzymes in Wild Type N. crassa

[0279] To better understand the processes by which filamentous fungi sense and respond to cellulose in their environment, next generation RNA sequencing techniques were used to profile genome-wide mRNA abundance in N. crassa. For these experiments, cultures grown for 16 hrs in sucrose minimal medium (SMM; in linear growth phase), and then shifted the culture from SMM to cellulose as a sole carbon source (CMM; cellulose minimal medium) were used; RNA samples were taken at 30 min, 1 hr, 2 hr and 4 hr following shift from SMM to CMM and compared to a culture shifted to SMM at identical time points. Typical patterns of expression for genes known to be associated with cellulose degradation are depicted in Figure 1A. This subset of genes increases in expression level approximately an order of magnitude within 30 min after transfer. Transcript abundance remains constant for approximately 1 hour before increasing by several more orders of magnitude between 2 and 4 hrs post transfer.

[0280] A very large number of genes change in expression profile following shift from SMM to CMM. Functional category analyses (Ruepp A et al, Nucleic Acids Res, 32: 5539-5545 (2004)) of this gene set revealed a large number are associated with the environmental stress response (Tian et al., Microbiology, 157: 747-759 (201 1). We therefore determined the transcriptional profile when 16 hr SMM-grown cultures were transferred to media with containing no carbon (NC) source. We observed that transcripts for a large number of genes (including many cellulases and hemicellulases) undergo the same initial increase in abundance (30 min-1 hr), but not the secondary increase (2-4 hrs). In cultures shifted to SMM, transcripts remain at or near their initial abundances, commonly increasing up to 2-fold by 4 hours, but remaining well below abundance levels seen in cellulose or no-carbon cultures. These results suggest that the first stage of transcript accumulation is a result of the lifting of carbon-catabolite repression and a general starvation response. The second stage is likely the result of a specific induction of transcription in response to the presence of cellulose. Results for several predicted cellulases and hemicellulases depicting these trends are shown in Figures IB and 1 C, respectively. The first stage of transcript accumulation is likely a result of the lifting of carbon- catabolite repression and a general starvation response. The second stage is likely the result of a specific induction of transcription in response to the presence of cellulose.

[0281] To identify genes regulated at the level of transcription by the presence of cellulose, transcript abundances between libraries from the three carbon source conditions (SMM, CMM and NC) at 1 hr (starvation response) and 4 hrs (cellulose-specific response) after transfer were compared. Results from CMM and SMM cultures were performed in biological triplicate for these analyses. Differentially expressed genes were identified as those that (a) showed statistically significant changes in abundance as estimated by the Cuffdiff software package with a 5% false discovery rate and (b) showed at least a two-fold change in abundance consistently across all replicates of each condition.

[0282] Figure 2 illustrates the abundance changes observed among these conditions. As many as 45% of predicted transcripts in the N. crassa genome (nearly 4500 genes) have altered abundance in CMM or NC cultures as compared to SMM culture, representative of the broad physiological changes that occur rapidly on transfer to carbon-poor conditions (Figs. 2A, 2B, 2D, and 2E). In contrast, at the one hour time point, a relatively small number of genes showed statistically differing transcript abundance in NC cultures compared to CMM cultures, with 410 genes showing differential expression, 276 of which are more highly expressed in the NC culture versus CMM (Fig. 2C). The majority (257) of these genes are more highly expressed in NC conditions than in SMM conditions and are therefore likely a general starvation response. Four hours after media transfer, a new collection of genes emerges in the far upper right of Figure 2D (4 hr) versus Figure 2A (1 hr) (CMM versus SMM) and especially in the upper portion of Figure 2F (4 hr) versus Figure 2C (1 hr) (CMM versus NC). These 552 differentially expressed genes depicted in Figure 2F comprise the cellulose transcriptional response (genes that either increase in expression level or decrease in expression level). This group is more specific to cellulose induction rather than a general response to starvation. Of particular interest are the 321 genes showing elevated expression level on cellulose, with abundances up to 4,000 times that of the no-carbon cultures. This induced group of genes includes 16 of the 21 predicted cellulases and 12 of the 19 predicted hemicellulases from N. crassa genome. Also included are 30 less well- characterized enzymes with predicted carbohydrate hydrolase, esterase or lipase activity with probable secretion signal peptides and 4 enzymes with predicted activity on disaccharides and signal peptides, as well as 44 hypothetical proteins with predicted signal peptides (Tables 1 A- IE; In Table 1A-1E, genes are indicated in the left side column; the listed genes are the same for each of Tables 1A-E. Tables 1A-1E contain different results relating to the same set of genes.). Cellulose also induces transcription of 21 genes with predicted roles in protein synthesis, modification and secretion as well as 8 predicted carbohydrate transporters, including recently characterized cellobiose transporters (Galazka et al., Science, 330: 84-86 (2010)). The resulting gene list includes approximately half of the genes identified in a similar study employing Bayesian analysis of microarray data (Tian et al., Proc Nat Acad Sci. USA, 106: 22157-22162, (2009)). Of those genes identified by Tian et al. that were not regulated by cellulose in our study, half were found to be differentially expressed under starvation and/or derepression conditions (Fig. 3).

Example 2: Essential Regulators for Cellulose Degradation

[0283] To identify transcription factors required for cellulose degradation, we screened the -200 N. crassa transcription factor deletion collection for mutants with deficient growth on cellulose. Two mutants were identified with severe growth defects on cellulose but normal growth on sucrose. The corresponding genes, NCU07705 and NCU08042, were provisionally named cdr- 1 and cdr-2, respectively for cellulose degradation regulator 1 and 2. However, it was later found that the "cdr" prefix was previously used for genes involved in cadmium resistance in N. crassa. Accordingly, the genes NCU07705 and NCU08042 were renamed as clr-1 and clr-2, respectively for cellulose degradation regulator 1 and 2. It should be noted that while some of the accompanying figures may refer to cdr-1 and cdr-2, the descriptions of these figures in Examples 2-4 below refer to cdr-1 as clr-1, and refer to cdr-2 as clr-2.

[0284] Deletion mutants for clr-1 and clr-2 exhibit little to no growth on cellulose, PASC or CMC in either liquid or solid culture, but exhibit wild type growth on minimal medium containing xylan (XMM), a hemicellulose, as a sole carbon source (Figs. 4A and 4B).

Furthermore, when clr mutants are grown on SMM and subsequently transferred to CMM, they are deficient for cellulase and hemicellulase activity and secretion, as well as total protein secretion. However, when transferred to xylan and allowed to grow for 24 hr, they exhibit normal hemicellulase enzyme activity and protein secretion (Figs. 4C-4F). These phenotypes were taken as evidence that clr-1 and clr-2 are essential transcription factors for the specific detection and metabolic response to the presence of cellulose.

[0285] clr-1 and clr-2 encode proteins that belong to the fungal specific zinc binuclear cluster superfamily. This large and diverse family of transcriptional regulators includes many previously described regulators of alternative carbon metabolism, including gal-4, ace- 1 , and xlnR (xyr-1) (Strieker et al., App. Micro Biotech., 78: 21 1-220 (2008)). Members of this family typically maintain two conserved domains, a zinc (2) cysteine (6) binuclear cluster coordinating DNA binding, and a conserved central domain roughly corresponding to what is known as the middle homology region (Campbell et al., Biochem. J., 414: 177- 187 (2008)). As shown in Figure 5A, clr-1 and clr-2 also contain the conserved zinc (2) cysteine (6) binuclear domain, as well as a conserved central PFAM04082 domain.

[0286] An examination of the expression patterns of clr-1 and clr-2 reveals potential differences in their regulation and mode of action. Both genes are essentially off under SMM conditions, however upon exposure to cellulose, clr-1 transcript levels increase within the first 30 minutes and then slowly increase throughout the 4 hr time point. Meanwhile, clr-2 expression levels remain low at 30 minutes, increases slightly by 1 hour, but doesn't dramatically increase until the 4 hr time point (Fig. 5C). Thus, clr-2 expression closely mimics the expression pattern of cellulolytic genes, and thus may undergo a similar de-repression stage.

[0287] clr-1 and clr-2 expression is also specific to cellulose. Exposure and growth in SMM, XMM and NC have little effect on clr-1 or clr-2 transcript levels when compared to cellulose (Fig. 5D). However, although abundance of clr-1 transcript under CMM conditions is relatively unaffected in the clr-2 deletion strain, the induction of clr-2 expression upon cellulose exposure is abolished in a clr-1 mutant. Thus, accumulation of clr-2 transcript requires both the expression of clr- 1 and the presence of the cellulose signal.

[0288] To determine whether mis-expression of clr-1 could induce clr-2 expression in the absence of cellulose, clr-1 was tagged with GFP and placed under the constitutively expressed promoter ccg-1 (Fig. 5B). The ccg-1 clr-1 -gfp construct was able to complement for the clr-1 knockout and showed localization of CLR-1 to the nucleus (Fig. 5E). When grown on CMM, RT-qPCR analysis shows wild type induction of clr-2 and the major cellulase NCU07340 (cbh- 1) in the ccg-1 clr-l -gfp strain (Fig. 5F). However, when the ccg-1 clr-l -gfp strain was grown in SMM, expression levels of cbh-1 and clr-2 remain the same as wild-type grown on in SMM (Fig. 5F). Thus, inappropriate expression of clr-1 in SMM is not sufficient to induce expression of clr-1 or the cellulolytic regulon. This result suggests that clr-1 is post-transcriptionally modified in order to induce clr-2 and cellulase expression.

Example 3: Phylogenetic Analysis

[0289] A phylogenetic analysis of clr-1 and clr-2 was conducted to gain insight on the evolutionary history of the two genes. Maximum likelihood phylogenetic trees of clr-1 and clr-2 homologs largely recapitulated previous fungal trees (Fig. 6). Trees created using Bayesian inference also shows congruent trees (Fig. 9). Similarities between the two trees lend support to the idea that CLR-1 and CLR-2 may be co-evolving and the hypothesis that they may act together as a heterocomplex.

Example 4: CLR-1 and CLR-2 Regulons

[0290] Strains containing deletions of clr- 1 or clr-2 have similar global expression profiles to a wild type strain when transferred from SMM to NC. Importantly, predicted cellulase genes have FPKMs that are similar both in magnitude and relation to each other in wild type NC culture as compared to Aclr-l or Aclr-2 CMM cultures (Fig. 7A). Thus wild type NC cultures and the Aclr- l or Aclr-2 CMM culture appear to undergo an identical starvation response. When compared to wild type on CMM, it is clear that both Aclr-l and Aclr-2 mutants failed to induce cellulase gene transcripts in response to exposure to cellulose and are therefore starving (Fig. 7A). Under CMM conditions, hemicellulase gene profiles were more mixed in the Aclr-l or Aclr-2 mutants, with transcripts from some predicted hemicellulase genes showing wild type abundance in the Aclr-l or Aclr-2 mutants, while others were dependent upon functional clr-1 and clr-2 for induction (Fig. 7B).

[0291] Global expression analyses in the Aclr-l or Aclr-2 mutants transferred to CMM revealed that they show differential expression of a smaller number of cellulose-specific genes identified in a wild type transferred to CMM (cellulose regulon) (Figs. 7C and 7D). These results indicate that the cellulose regulon includes genes regulated by clr-1 and/or clr-2 as well as some genes under the regulation of an independent mechanism. To delineate the respective regulons, all genes exhibiting differential expression in CMM versus NC conditions or in wild type versus the deletion mutants on CMM were hierarchically clustered by their FPKM values. The resulting clusters indicate that clr-1 and clr-2 share a common regulon that is a major subset of the cellulose induced genes (Fig. 7E). Of the 321 genes that increase in expression level identified in the wild type cellulose regulon (see above), clr-1 and clr-2 are essential for the induction of 204 genes. A further 59 genes required functional clr-1 and clr-2 for increased expression levels, in comparison to wild type. Importantly, the clr-1 and clr-2 regulons almost completely overlap each other (clr regulon). The clr regulon is highly enriched for genes encoding cellulases, polysaccharide active enzymes, transporters and protein synthesis and secretion components with respect to the total cellulose regulon (Fig. 7F). Some, but not all predicted hemicellulases are also under clr regulation. Predicted hemicellulases under clr regulation increase in to a higher expression level after transfer to CMM than transfer to XMM. This cellulase-like expression pattern may indicate that these genes actually encode cellulose active enzymes, or that is advantageous to maintain a group of true hemicellulases under tight co-regulation with cellulases. It should be noted that fungi never encounter pure cellulose without hemicellulose under natural settings.

Example 5: Dependence of Cellobiose Induction on CLR-1 and CLR-2

[0292] When fungal cellulases interact with cellulose, cellobiose and glucose are the main soluble products. Without wishing to be bound by theory, it is believed that cellobiose, or a product derived therefrom, is the inducing molecules for fungal cellulases. However, to be utilized, the cellobiose must be hydrolyzed to glucose by beta-glucosidase enzymes. In cultures with pure cellobiose, glucose concentrations quickly rise and cellulase induction is blocked through carbon catobolite repression. Moreover, glucose repression of cellulases is abolished in N. crassa strains in which the most highly expressed beta-glucosidase genes (NCU00130, NCU08755 and NCU04952) are deleted (Znameroski et al., Proc Natl Acad Sci U S A. 2012 Apr 17; 109(16):6012-7). This mutant system allows for very specific cellulase induction

experiments free of any other signaling molecules that may contaminate Avicel® (crystalline cellulose -98-99% pure), which is purified from natural plant cell wall material (with ~1 -2% hemicellulose contamination).

[0293] We generated N. crassa mutant strains carrying deletions for the beta-glucosidase genes and for clr-1 or clr-2. When these ABG+Aclr mutants were switched from sucrose to cellobiose, the major cellulase cbh-lwas not induced (Fig. 10). These results strongly suggest that cellobiose, or a product derived therefrom, is the signal molecule that activates the clr-l/clr- 2 pathway.

[0294] In contrast to cbh-1 , the cellodextrin transporter cdt-2 is still strongly induced in the ABG Aclr-2 mutant. This difference in regulation of cellulase genes and cellobiose utilization genes by clr-1 and clr-2 is consistent with the disclosed model network in which clr-1 is intimately involved in cellobiose detection and utilization but clr-2 only regulates cellulase genes and their secretion (Fig. 8).

Example 6: Effect of Mis-Expression of CLR-1 on Cellulase Expression

[0295] Without wishing to be bound by theory, it is believed that clr-1 undergoes post- transcriptional modification or activation in the presence of cellobiose and the absence of repressing carbon sources. Consistent with this belief, it was shown that merely forcing transcription of clr-1 under non-inducing conditions did not result in cellulase production.

[0296] We generated a N. crassa strain with a GFP tagged copy of clr-1 under control of the ccg-1 promoter at the his-3 locus. The ccg- 1 is responsive to the N. crassa circadian rhythm and nutrient conditions. For the purposes of these experiments, the ccg-1 promoter served as a constitutive promoter, driving greater clr-1 transcription that is normally seen under rich carbon (sucrose) or starvation conditions and lower clr- 1 transcription under Avicel® conditions as seen from the native promoter.

[0297] Our clr-1 mis-expression strain produces no detectable cellulase activity in sucrose culture. Results from a CMCase enzyme activity experiment are show in Figure 1 1 A. The results show that the enzyme activity was lower in the mis-expression mutant than in the wild- type strain (Fig. 1 1 A). The mis-expressed clr-1 in this strain was tagged with a C-terminal GFP marker that may reduce its transcriptional efficiently. However, this reduced activity is sufficient for growth on Avicel®.

[0298] Further, transcription of the major cellulase cbh-1 showed very poor correlation to clr-1 transcription in both wild type and mutant strains (Fig. 1 IB). Transcription of cbh-1 correlated with the presence or absence of a cellulase induction by Avicel®, but was not correlated with clr-1 expression levels. Example 7: Effect of Mis-Expression of CLR-2 on Cellulase Expression and Activity Under Non-Inducing Conditions

[0299] We generated N. crassa strains expressing clr-2 under control of the ccg-1 promoter at the his-3 locus. Expression of clr-2 under non-inducing conditions was sufficient to induce cellulase gene expression and activity (Fig. 12A). Regardless of media condition, transcript abundance of cbh- 1 was directly proportional to clr-2 transcript abundance (Fig. 12 A). This proportionality was not dependent on either inducer or a functional copy of clr-1. However, cbh- 1 induction was most efficient in the presence of both inducer and a functional copy of clr-1.

[0300] Transcriptional induction of cellulase genes by clr-2 mis-expression resulted in secretion of active cellulases (Fig. 12B). Figure 12B shows the results of a CMCase enzyme activity experiment with wild-type (WT) and clr-2 mis-expression strains. The sucrose grown mis-expression strain quickly developed enzymatic activity comparable to that of Avicel© grown WT strains (Fig. 12B).

[0301] Consistent with observations that clr-2 transcript abundance and cbh-1 transcript abundance are correlated, mis-expression strains with higher expression levels of clr-2 secreted more protein with greater enzyme activity (Figs. 12C and 12D). Figures 12C and 12D show CMCase enzyme activity and secreted protein from clr-2 mis-expression strains pre-grown in sucrose and shifted to either Avicel© or sucrose media. In the Δ/Pccgl -clr-2 strain, clr-2 is deleted from its native locus and expressed under control of the ccg- 1 promoter at the his-3 locus. In the Native/Pccgl -clr-2 strain, the native copy of clr-2 is retained in addition to the ccg- 1 driven copy of clr-2 at the his-3 locus.

[0302] Additionally, an SDS-PAGE gel of culture supernatants indicated that the clr-2 mis- expression strain secretes a similar spectrum of enzymes on sucrose as does the WT strain on Avicel© (Fig. 13 A). RNAseq analyses of major cellulase transcripts after a media shift from sucrose to no carbon conditions confirmed that enzymes in the clr-2 mis-expression strain were induced to similar levels as in the WT shifted to Avicel® (Fig. 13B).

[0303] RNAseq results from the clr-2 mis-expression strain complimented results from a wild-type (WT) N. crassa strain in various media conditions. The clr deletion strains on Avicel© and the ABG mutants on cellobiose illustrate several modes of transcriptional induction of WT strains on Avicel®. Figure 14 shows hierarchical clusters of the approximately 200 genes induced by Avicel® in these strains and conditions. Of these genes, approximately one quarter are not induced by cellobiose, but are induced by hemicellulosic contamination of Avicel®, including several hemicellulase and pentose sugar utilization genes (Fig. 14). Of the cellobiose induced genes, all showed some decrease in abundance in the Aclr-l and Aclr-2 strains on Avicel® and approximately 2/3 were dependent on clr-1 and/or clr-2 (Fig. 14). These genes showed a no carbon-like expression profile in the clr-1 and clr-2 deletion strains on Avicel®. Most of the clr-dependent genes were strongly induced in the clr-2 mis-expression mutant (Fig.

14) . One cluster of approximately 50 genes had complex expression patterns indicating some level of modulation of expression by clr-1 and/or clr-2. Among clr-modulated genes, most were more strongly affected in the Aclr-l deletion strains and had little to no induction in the clr-2 deletion strain. Notable among clr-modulated genes most strongly affected by clr-1 are several genes involved in cellobiose utilization.

Example 8: Condition-Specific Post Translational Modification of CLR-1

[0304] Without wishing to be bound by theory, it is believed that one way that clr-1 may be activated in response to cellobiose and/or global metabolic state is through post-translational modification. Western blot analysis of V5-tagged clr-1 at its native locus indicated a small but detectable shift in the mature CLR-1 protein when cultures were shifted from sucrose to Avicel® or no carbon conditions (Fig. 15). As shown in Figure 15, the CLR-1 protein, which is predicted to be 78 kDa, ran in two bands on the gel. The larger band was more abundant in culture shifted to sucrose, cellobiose, xylan and xylose; whereas the smaller band was more abundant in Avicel® and no carbon conditions (Fig. 15). These results suggest that CLR-1 undergoes modification or selective degradation under starvation conditions. Moreover, while clr-1 transcript abundance was much higher under Avicel® conditions than under sucrose conditions, there were comparable amounts of mature CLR-1 protein under both of these conditions (Fig.

15) . Without wishing to be bound by theory, it is believed that these results suggest that there is increased turnover under starvation conditions.

Example 9: Identification of Direct Targets of CLR-1 and CLR-2

[0305] To further characterize the CLR regulons and their DNA binding motifs, chromatin immunoprecipitation (ChIP) was conducted on epitope-tagged CLR-1 and CLR-2 proteins. The experimental setup was similar to the RNAseq media swaps, with N. crassa strains grown on minimal media with sucrose for 16 hours then switched to Avicel® for 24 hours. For these experiments, CLR-1 was GFP tagged and under the control of the ccg-1 promoter, and CLR-2 was mCherry tagged and also under the ccg-1 promoter. The subsequent libraries yielded approximately 417 target genes in the CLR-1 ChlPseq library and 318 genes in the CLR-2 library (Fig. 16A).

[0306] In order to determine whether CLR-1 and CLR-2 are able to directly control the expression of genes upregulated on cellulose, we compared their ChlP-Seq regulons to the wild- type RNA-Seq regulon containing the 212 genes upregulated on cellulose (Fig. 13B). CLR-1 and CLR-2 together or separately bound to the promoter regions of approximately half the genes induced on Avicel® (Fig. 16A). The CLR proteins did not bind the promoters of genes down- regulated (over 2-fold down) on Avicel® versus no-carbon. These results indicate that CLR-1 and CLR-2 function strictly as transcriptional activators.

[0307] Overall, the ChlP-Seq results (Fig. 16A) largely recapitulated the RNA-Seq results (Fig. 13B). The CLR-1 and CLR-2 proteins together bound 40 genes that included a core set of 10 of the most highly expressed cellulase genes along with 2 hemicellulase genes. Additional genes of note within the 40 gene set included xlr-1 , a regulator of hemicellulase expression, vib- 1 which is involved in secretion, and NCU03184 (flbC) which has reduced growth on Avicel® when deleted.

[0308] The large set of CLR-1 -bound genes that that are not within the Avicel®/cellulose regulon have enriched functional gene categories that are predicted to be involved with interaction with the environment and signaling (287 gene set, Fig. 16A). These results are consistent with the belief that CLR-1 is specifically involved in sensing of cellobiose in the environment (Fig. 8). The gene set bound only by CLR-2 and not within the Avicel®/cellulose regulon was not enriched for any functional category (173 gene set; Fig. 16A).

[0309] The CLR-1 protein was also found to be bound at the promoters of both the clr-1 and clr-2 genes (Fig. 16B). CLR-1 binding can be seen throughout the clr-2 promoter region including through the annotated hypothetical gene NCU l 1779 (Fig. 16B). However, as NCUl 1779 is not expressed in the 200 plus RNA-Seq experiments under a wide variety of conditions, we do not believe that NCUl 1779 is a protein-encoding gene. These results suggest that CLR-1 binding at the clr-1 and clr-2 promoters provides a positive feedback loop for clr-1 expression and verifies clr-2 as a downstream target of CLR-1.

[0310] Although CLR-1 binds to a large number of cellulose responsive genes, it does not appear to bind to cellulose degrading enzymes by itself; as CLR-2 was always found bound in an adjacent region of these promoters. These results support the hypothesis that CLR-2 is the main activator of cellulases and can drive their expression alone when mis-expressed (Fig. 12). In addition, almost all promoter regions bound by both CLR-1 and CLR-2 overlapped with each other. This result supports the hypothesis that CLR- 1 and CLR-2 interact physically at these promoters (Fig. 16C).

Example 10: Conservation of CLR Protein Sequences and Function in Filamentous Ascomycete Fungi

[0311] To assess whether clr-1 and clr-2 homologs function to regulate genes involved in plant cell-wall deconstruction in other filamentous ascomycete species, we generated clr-1 and clr-2 homolog deletion strains in the distantly related fungus Aspergillus nidulans in AN5808 (clrA) and AN3369 (clrB). Similar to N. crassa Aclr-l and Aclr-2 mutants, the A. nidulans AclrA and AclrB deletion strains were deficient for cellulase and xylanase activity, as well as total protein secretion when pre-grown glucose cultures were transferred to Avicel® (Fig. 17).

Enzyme activity was abolished in the AclrB mutant, but the AclrA mutant showed ~50% of wild-type (WT) activity (Figs. 17A and 17B). Both deletion mutants were deficient for growth on cellobiose, although AclrB was more strongly affected (Fig. 17C). Consistent with enzyme data, the induction pattern of major cellulase genes in the AclrB mutant was several thousandfold less than WT (Fig. 17D). However, in the AclrA mutant the average induction was two- to four-fold less. On a per-gene basis, this decrease was not statistically significant (P < 0.05) for three of four tested cellulases (P = 0.049, 0.052, 0.105, and 0.121 for AN1273, AN7230, AN0494, and AN5175, respectively), but considering all of the genes together, the null hypothesis that AclrA has WT levels of cellulase gene expression was not supported. These results support the conclusion that clrA has a less important role in cellulase induction in A. nidulans compared with clr-1 in N. crassa. However, the function of CLR-2/ClrB as an essential activator for cellulase gene expression and activity is conserved between N. crassa and A.

nidulans, two of the most widely divergent species of filamentous ascomycete fungi.

[0312] Results from RT-PCR analysis showed that the Avicel®-induced expression of clrA on Avicel® was dependent on the presence of clrB, but not vice versa (Fig. 17E). This result suggests that the growth defect of AclrB on cellobiose could be an additive effect of reduced expression of clrA and other genes.

Example 11: Mis-Expression of CLRA and CLRB in Aspergillus nidulans [0313] Given the results showing the conservation between clrB and clr-2 as essential factors for cellulase gene expression in both N. crassa and A. nidulans (Fig. 17) and that mis-expression of clr-2 is sufficient to induce cellulase expression under non-inducing conditions in N. crassa (Fig. 12), we decided to test whether mis-expression of clrB in A. nidulans can induce cellulase expression. In a AclrB A. nidulans strain, the clrB gene was put under the control of gpdA promoter and integrated into the genome at the pyrG locus of the AclrB strain, with the A.

fumigatus pyroA gene as a selective marker. Figure 18A shows that the expression of clrB mRNA in the clrB mis-expression strain was much higher than in the wild-type strain in all conditions tested (glucose, no carbon and Avicel®). As shown in Figures 1 8B and 1 8C, the mis- expression of clrB restored expression of cbhD on Avicel® and the strain grew as well as wild- type on cellobiose. These results suggest that the mis-expressed ClrB protein is functional. Although the clrB mis-expression strain exhibited a higher CMCase activity than wild-type after growth on cellobiose for 48 hrs (Fig. 18C), no CMCase activity was detected in the clrB mis- expression strain grown on glucose. Moreover, the high mRNA level of clrB in the clrB mis- expression strain on Avicel® did not lead to higher mRNA level of cbhD at 6 hrs (Figs. 18A and 18B).

Example 12: Expression of CLRA and CLRB in Neurospora crassa

[0314] Considering the relatively high amino acid sequence similarity of CLR proteins in A. nidulans and N. crassa (49% identity between clr-1 and clrA, and 32% identity between clr-2 and clrB), we tested whether clrA and clrB could substitute for their homologs in N. crassa. A N. crassa Aclr-l strain expressing clrA under the ccg-1 promoter was generated. The Aclr-l strain expressing clrA retained the severe growth defect on Avicel®, although it accumulated a similar amount of biomass as compared to wild-type on cellobiose (Figs. l 9A and 19B). A N. crassa Aclr-2 strain expressing clrB under the ccg-1 promoter was also generated. Although clrB is essential for growth on cellobiose and cellulase gene expression in A. nidulans, the mis- expressed clrB did not rescue the growth of N. crassa Aclr-2 on either Avicel® or cellobiose (Figs. 19C and ! 9D). These results suggest that the function of clr-1 /clrA in the cellobiose utilization pathway is conserved between A. nidulans and N. crassa, but the function of clr- 1/clrA and clr-2/clrB in the regulation of Avicel®-specific response may be divergent.

Example 13: DNA-Binding Motifs of N. crassa CLR Proteins

Clr-1 [0315] The top 50 CLR-1 chromatin-immunoprecipitation peaks, which were identified by sequence analysis (ChlP-Seq; promoter regions most frequently immunoprecipitated by antibody to epitope-tagged CLR-1), were searched for a characteristic DNA binding motif. The peaks were searched using the program MEME (Multiple Em for Motif Elicitation) and resulted in the motif depicted in Figure 20A, for a consensus binding site for CLR-1 in promoters of target genes. This motif has the characteristic inverted CGG repeats that is commonly found in this class of transcription factors. One of the important characteristics of the CGG inverted repeat is the spacing between them, which helps determine which transcription factors can bind to the location. The CLR-1 motif is separated by a single non-conserved nucleotide. This spacing has been seen in other transcription factors, but none with a related function.

Clr-2

[0316] The top CLR-2 chromatin-immunoprecipitation peaks, which were identified by sequence analysis (ChlP-Seq; promoter regions most frequently immunoprecipitated by antibody to epitope-tagged CLR-2), were searched for a characteristic DNA binding motif. The peaks were searched using the program MEME (Multiple Em for Motif Elicitation) and resulted in the motif depicted in Figure 20B, for a consensus binding site for CLR-2 in promoters of target genes. This motif has the characteristic inverted CGG repeats that is commonly found in this class of transcription factors. One of the important characteristics of the CGG inverted repeat is the spacing between them, which helps determine which transcription factors can bind to the location. The CLR-2 motif is separated by 1 1 non-conserved nucleotides. This spacing is the same as for the Saccharomyces cerevisiae Gal4 motif, the closest yeast homolog to CLR-2. There are 50 motif binding sites within the CLR-2 ChlP regulon with the predicted DNA binding motif, this number was increased to 84 with the simplified version of CCG(N1 1)CGG.

Example 14: CLR Protein Sequence Analysis

Clr-1

[0317] The N. crassa clr-1 amino acid sequence was aligned with 22 other clr-1 homologs to identify conserved motif sequences (Fig. 21 ). Sequences were aligned with the MAFFT alignment algorithm (available from the CBRC mafft website). Alignments were manually inspected for regions of conservation outside of known conserved domains in likely orthologs (as determined by phylogenetic analysis), but which were not well conserved in the nearest non-clr-1 paralogs in N. crassa and A. nidulans. The consensus sequence was determined with the Jalview software suite.

[0318] As shown in Figure 21 , the sequence alignment identified the zinc(2)-cysteine(6) binuclear cluster domain, which is conserved in members of the fungal specific zinc binuclear cluster superfamily, at amino acids 220-275 of the consensus sequence shown at the bottom of the figure. The conserved zinc(2)-cysteine(6) binuclear cluster domain had the following sequence: C-E-V-C-R-S-R-K-S-R-C-D-G-T-K-P-K-C-K-L-C-T-E-L-G-A-E-C-I-Y-R-E (SEQ ID NO: 235).

[0319] The sequence alignment also identified the fungal -specific transcription factor

PFAM04082 conserved central domain at amino acids 435-760 of the consensus sequence (Fig.

21). The PFAM04082 transcription factor domain had the following sequence: I-E-A-Y-F-E-R-

V-N-V-W-Y-A-C-V-N-P-Y-T-W-R-S-H-Y-R-T-A-L-S-N-G-F-R-E-G-P-E-S-C-I-V-L-L-V-L-

A-L-G-Q-A-S-L-R-G-S-I-S-R-I-V-P-X-E-D-P-P-G-L-Q-Y-F-T-A-A-W-X-L-L-P-G-M-M-T-X-

N-S-V-L-A-A-Q-C-H-L-L-A-A-A-Y-L-F-Y-L-V-R-P-L-E-A-W-N-L-L-C-T-T-S-T-K-L-Q-L-L-

L-M-A-P-N-R-V-P-P-X-Q-R-E-L-S-E-R-l-Y-W-N-A-L-L-F-E-S-D-L-L-A-E-L-D-L-P-H-S-G-

I-V-Q-F-E-E-N-V-G-L-P-G-G-F-E-G-E-E-D-E-X-D-E-E-A-D-X-D-Q-E-I-A-X-V-T-A-V-G-R-

D-E-L-W-Y-F-L-A-E-I-A-L-R-R-L-L-N-R-V-S-Q-L-I-Y-S-K-D-T-P-Y-S-K-G-P-S-M-A-S-T-

T-S-L-E-P-I-V-A-E-L-D-F-Q-L-T-Q-W-Y-E (SEQ ID NO: 237), where X can be any amino acid residue.

[0320] Additionally, the sequence alignment identified five conserved sequence motifs that can be used to identify clr-1 transcription factors (Fig. 21 ). The first conserved motif was identified at amino acids 258-274 of the consensus sequence and has the following sequence: A- G-D-[KR]-[LM]-I-[LI]-[ED]-[RKQH]-L-N-R-I-E-[SNG]-L-L (SEQ ID NO: 188). The second conserved motif was identified at amino acids 851 -867 of the consensus sequence and has the following sequence: H-[HR]-[ADE]-G-H-[MLI]-P-Y-[IL]-[WF]-Q-G-A-L-S-[MI]-[VMI] (SEQ ID : 189). The third conserved motif was identified at amino acidsl66-l 80 of the consensus sequence and has the following sequence: [NP]-[PS]-[LKTS]-K-[RK]-[RK]-[NSP]-[TSN]- [EDST]-X-X-[VIAT]-[DE]-Y-P (SEQ ID NO: 190), where X can be any amino acid residue. The fourth conserved motif was identified at amino acids 330-340 of the consensus sequence and has the following sequence: G-G-[FLIS]-G-[TSG]-[WAH]-X-W-P-[PA]-[TS] (SEQ ID NO: 191). The fifth conserved motif was identified at amino acids 104-1 1 1 of the consensus sequence and has the following sequence: R-[NH]-[LM]-[ST]-[QP]-[STP]-[SP]-[DE] (SEQ ID NO: 192).

Clr-2

[0321] The N. crassa clr-2 amino acid sequence was aligned with 21 other clr-2 homolgs to identify conserved motif sequences (Fig. 22). Sequences were aligned with the MAFFT alignment algorithm (available from the CBRC mafft website). Alignments were manually inspected for regions of conservation outside of known conserved domains in likely orthologs (as determined by phylogenetic analysis), but which were not well conserved in the nearest non-clr-1 paralogs in N. crassa and A. nidulans. The consensus sequence was determined with the Jalview software suite.

[0322] As shown in Figure 22, the sequence alignment identified the zinc(2)-cysteine(6) binuclear cluster domain, which is conserved in members of the fungal specific zinc binuclear cluster superfamily, at amino acids 65-1 10 of the consensus sequence shown at the bottom of the figure. The conserved zinc(2)-cysteine(6) binuclear cluster domain had the following sequence: C-A-E-C-R-R-R-K-I-R-C-D-G-E-Q-PC-G-Q-C-X-W-Y-X-K-P-K-R-C-F-Y-R-V-X-P-S-R-K (SEQ ID NO: 236), where X can be any amino acid residue.

[0323] The sequence alignment also identified the fungal-specific transcription factor PFAM04082 conserved central domain at amino acids 368-555 of the consensus sequence (Fig. 22). The PFAM04082 transcription factor domain had the following sequence: I-D-A-Y-F-K-R- V-H-X-F-X-P-M-L-D-E-X-T-F-R-A-T-Y-L-E-G-Q-R-K-D-A-P-W-L-A-L-L-N-M-V-F-A-L-G- S-I-A-A-M-K-S-D-D-Y-N-H-X-X-Y-Y-N-R-A-M-E-H-L-X-L-D-S-F-G-S-S-H-X-E-T-V-Q-A- L-A-L-M-G-G-Y-Y-L-H-Y-l-N-R-P-N-X-A-N-A-L-M-G-A-A-L-R-M-A-S-A-L-G-L-H-R-E-S- L-A-Q-X-X-A-S-S-Q-K-G-V-N-X-S-D-X-A-S-A-E-T-R-R-R-T-W-W-S-L-F-C-L-D-T-W-A-T- T-T-L-G-R-P-S-X-G-R-W-G (SEQ ID NO: 238), where X can be any amino acid residue.

[0324] Additionally, the sequence alignment identified four conserved sequence motifs that can be used to identify clr-2 transcription factors (Fig. 22). The first conserved motif was identified at amino acids 140-152 of the consensus sequence and has the following sequence: [VL]-[ED]-[KAE]-L-S-[QTSN]-[STN]-[LVI]-[DE]-[DE]-[YC]-[RK]-[STV] (SEQ ID NO: 184). The second conserved motif was identified at amino acids 800-818 of the consensus sequence and has the following sequence: [MLI]-[STI]-G-W-N-A-V-W-[FLW]-[IVLCT]-[FY]-Q-[AS]-X- [ML]-[VI]-P-L-[ILV] (SEQ ID: 185), where X can be any amino acid residue. The third conserved motif was identified at amino acids614-619 of the consensus sequence and has the following sequence: [ED]-X-L-[AV]-[AVI]-[STAL] (SEQ ID NO: 186), where X can be any amino acid residue. The fourth conserved motif was identified at amino acids 14-19 of the consensus sequence and has the following sequence: M-[FY]-[HIL]-T-F-[QE] (SEQ ID NO: 187).

Materials & Methods for Examples 1-14 Include:

Strains

[0325] The wild-type reference strain and background for all N. crassa mutant strains was FGSC 2489 (Neurospora crassa 74-OR23-1 V A). Deletion strains for clr-1 and clr-2 with their open reading frames replaced by a hygromycin resistance cassette (FGSC 1 1029 and FGSC 15835 respectively) were obtained from the Fungal Genetics Stock Center at the University of Missouri, Kansas City, MO. The wild-type A. nidulans reference strain was FGSC 4A. Gene deletions in A. nidulans were carried out by transforming FGSC A l 149 (pyrG89; pyroA4;

nkuA::argB) with knockout cassettes obtained from the Fungal Genetics Stock Center at the University of Missouri, Kansas City, MO.

[0326] Transformants were crossed to L01496 (fwAl , pyrG89, nicA2, pabaAl , from Berl R. Oakley Department of Molecular Biosciences, University of Kansas, Lawrence, KS) to remove nkuA::argB and pyroA4.

Culture Condit ions for Media Shift Assay

[0327] N. crassa strains were inoculated into 3 mL agar slants with Vogel 's minimal media (2% sucrose as carbon source; SMM) and grown at 30°C in the dark for 48 hours, then at 25°C in constant light for 4-10 days to stimulate conidia production. Suspended conidia were then inoculated into 100 mL of Vogel's minimal media at 106 conidia/mL and grown 16 hours at 25°C in constant light and agitation. The mycelial cultures were then centrifuged at 3400 rpm for 10 min at room temperature and washed with Vogel's minimal media (VMM) without a carbon source. Washed mycelia were re-suspended in 100 mL Vogel 's with 2% carbon source (cellulose or hemicellulose). The cellulose used in all experiments was Avicel© PH-101 (Sigma Aldrich, MO). The model hemicellulose used was Beechwood Xylan (Sigma Aldrich, MO). [0328] A. nidulans cultures were grown on minimal media (MM). Carbon sources were 1 % wt/vol unless otherwise noted. Conidia were inoculated into 100 mL liquid media at 4 ^χ 106 conidia/mL and grown at 37°C in constant light and shaking (200 rpm). A. nidulans cultures were grown 16-17 hr on MM-glucose. A 15 mL sample was taken at time 0. The remaining culture was filtered through miracloth, washed, and transferred to 100 mL MM containing 1% Avicel®. RNA was extracted as above and mRNA abundance was compared between the 8 hr and time 0 samples by quantitative RT-PCR. Fold-induction was calculated as the ratio of the mRNA level normalized to act A at 8 h vs. act A at time 0. For enzyme activity assays, culture supernatants were sampled at 48-120 hr, centrifuged at 2,390 ^χ g twice to remove mycelia and stored at 4°C for analysis.

[0329] For RNA expression profiling, cultures were sampled post-transfer at 30 minutes, 1 hr, 2 hrs and 4 hrs. Mycelia samples were collected by filtering onto WHATMAN (TM) paper and were immediately flash-frozen in liquid nitrogen. Total RNA was extracted as described in (Kasuga et al., Nucleic Acids Res., 33: 6469-6485 (2005)).

[0330] For enzyme activity assays, culture supernatants were sampled at 24 hours, filtered with WHATMAN (TM) paper and stored at 4°C for analysis within 72 hrs.

RNA sequencing

[0331] RNA samples were reverse transcribed and prepared for high throughput sequencing with protocols adapted from lllumina Inc. Briefly, mRNA was purified with DYNABEADS (TM) Oligo dT magnetic beads (lllumina). Purified mRNA was fragmented with buffered zinc solution from Ambion (Cat # AM8740). First and second strand cDNA synthesis was carried out using Superscript II Reverse Transcriptase (Invitrogen) and DNA pol I (Invitrogen) and random primers. lllumina sequencing adapters were then ligated to the cDNA, 200bp fragments were purified by gel electrophoresis, and PCR enriched with Pfx DNA polymerase (Invitrogen). Libraries were sequenced on the HiSeq 2000 DNA sequencing platform at the Vincent J. Coates Genomics Sequencing Laboratory at the California Institute for Quantitative Biosciences, Berkeley CA. Approximately 60 million single end 50 base-pair reads were obtained per library.

Analysis of Differential Expression [0332] To establish biological variation, triplicate cultures were sampled and analyzed for the wild type strain on cellulose and sucrose at 1 hour and 4 hours after the media shift. For all other strains and conditions, a single NAseq library was analyzed.

[0333] Sequenced libraries were mapped against predicted transcripts from the N. crassa OR74A genome (version 10) with Bowtie (Langmeadet al., (2009) Genome Biol 10:R25). Transcript abundance was estimated with Cufflinks using upper quartile normalization and mapping against reference isoforms from the Broad Institute. Genes exhibiting statistically significant expression changes between strains or growth conditions were identified with Cuffdiff, using upper quartile normalization and a minimum raw count of 5 reads (Roberts A, Trapnell et al., (201 1) Genome Biology. 12:R22). The genes identified by Cuffdiff were then filtered to select only those exhibiting a two-fold change in estimated abundance between all biological replicates of each strain/condition tested and only those genes with an FPKM consistently above 5 in at least one strain/condition were considered significant.

[0334] To compare genes exhibiting altered expression in clr mutants to those exhibiting altered expression in response to cellulose, genes were hierarchically clustered by their FPKMs in the wild type strain on cellulose, wild type on no-carbon and in the Aclr-l and Aclr-2 strains on cellulose, all at 4 hours after media shift. Prior to clustering, FPMKs were log transformed, normalized across strains/conditions on a per-gene basis and centered on the median value across strains/conditions.

Enzyme Activity Assays

[0335] To assess total cellulase activity, 500 μΐ_^ of culture supernatant was incubated with 2.5 mg cellulose in 500 μΐ, of 100 mM sodium acetate, pH 5 for 5 hours at 37° C. 40 \iL of incubated sample was then added to 160 μΐ, assays solution containing dianisidine, peroxidase and glucose oxidase. In this assay, hydrogen peroxide released by glucose oxidation then oxidizes the dianisidine resulting in a color change proportional to glucose concentration.

Absorbance of the glucose assays were read at 540 nm in a VERSAmax microplate reader (Molecular Devices). The background was subtracted with a no-cellulose control reaction and compared to that of glucose standards.

[0336] To assess hemicellulase activity, 100 μΐ, of culture supernatant was incubated with 9 mg xylan in 900 \L of 100 mM sodium acetate, pH 5 for 30 minutes at 50° C. Released xylose was then measured by reduction of 3, 5-dinitrosalicylic acid in a similar manner as the glucose oxidase assay.

[0337] Total protein was determined with the Bradford assay (BioRad). Phylogenetic Analysis

[0338] Putative homologues to CLR-1 and CLR-2 were first identified through BLASTs to the NCBI protein database. The top hits from each BLAST were selected and were separately blasted to the Neurospora crassa protein database to verify CLR-1 or CLR-2 as the top hit and no other closely related Neurospora proteins. The phylogenetic trees were created using the maximum likelihood program PhyML with ALRT branch support. The CLR-2 tree has a loglk of -28588 and the CLR-1 tree has a loglk of -19403 (Anisimova M and Gascuel O, Systematic Biology, 55(4), 539-552 (2006)).

[0339] The phylogenetic trees were also run using Bayesian inference (MrBayes).

(Huelsenbeck et al., (2001) Science 294: 2310-2314). One million generations were run with 8 chains, trees were sampled every 100 generations, with a burn-in of 2,500. The CLR-1 tree converged to 0.0056 and the CLR-2 tree converged to 0.0027. The resulting trees showed congruency with those generated with maximum likelihood (Fig. 9 - phylogenetic trees based on Bayesian inference. They are identical to the maximum likelihood trees.)

RT-qPCR

[0340] Primers: clr-l-F 5 ' -ATG ACGCCG AACCGAGTG-3 ' (SEQ ID NO: 7)

clr-l-R 5 ' -C A AC A AC ACC AGA ATGCGG-3 ' (SEQ ID NO: 8)

clr-2-F 5'-TCCCGGCCATCAGACAGA-3' (SEQ ID NO: 9)

clr-2-R 5 ' -ATCGGC ACGGA AGGTTGTT-3 ' (SEQ ID NO: 10)

5-ac///j- 5'-TGATCTTACCGACTACCT-3' (SEQ ID NO: 1 1)

B-actin-R 5 ' -CAGAGCTTCTCCTTGATG-3 ' (SEQ ID NO: 12)

cbhl-F 5 ' -ATCTGGGAAGCGAACA AAG-3 ' (SEQ ID NO: 13)

cbhl-R 5 ' -TAGCGGTCGTCGGAATAG-3 ' (SEQ ID NO: 14)

[0341] Primer efficiencies were tested on a gDNA dilutions series to determine if they were comparable to each other. The 2489 wild type strain and the ccg-1 ::clr-l -GFP were grown on Vogels media with sucrose for 16 hours. The cultures were rinsed as described above and transferred to fresh media containing either Avicel®® or sucrose as the carbon source. RNA was extracted four hours post transfer. RT qPCR was carried out using the One Step Green ER kit (Invitrogen). One nanogram of total RNA was used in each RT-qPCR reaction and amplification conditions used were as described in the manufacturer's manual. Three technical triplicates were run for each sample. For the analysis, reactions were averaged and normalized to B-actin expression using the delta-delta Ct method (Livak KJ and Schmittgen TD, Methods, 25: 402 (2001)).

TABLES

Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU02704 branched-chain alpha-keto acid Amino Acid No

dehydrogenase E2 Metabolism Repression

NCU02727 glycine cleavage system T protein Amino Acid No

Metabolism Repression

NCU02936 proline oxidase Amino Acid No

Metabolism Repression

NCU03076 deIta-l-pyrroline-5-carboxylate Amino Acid No

dehydrogenase Metabolism Repression

NCU03415 aldehyde dehydrogenase Amino Acid No

Metabolism Repression

NCU03648 glutaminase A Amino Acid No

Metabolism Repression

NCU03913 2-oxoisovalerate dehydrogenase Amino Acid No

beta subunit Metabolism Repression

NCU05499 homogentisate 1,2-dioxygenase Amino Acid No

Metabolism Repression

NCU05537 fumarylacetoacetase Amino Acid No

Metabolism Repression

NCU05977 dihydrodipicolinate synthase Amino Acid No

Metabolism Repression

NCU06448 enoyl-CoA hydratase Amino Acid No

Metabolism Repression

NCU06543 acyl-CoA dehydrogenase Amino Acid No

Metabolism Repression

NCU07153 glutamate carboxypeptidase Amino Acid No

Metabolism Repression

NCU08216 cystathionine beta-synthase Amino Acid No

Metabolism Repression

NCU09116 aromatic aminotransferase Aro8 Amino Acid No

Metabolism Repression

NCU09266 methylmalonate-semialdehyde Amino Acid No

dehydrogenase Metabolism Repression

NCU09864 2-oxoisovalerate dehydrogenase Amino Acid No

alpha subunit Metabolism Repression

NCU11195 D-isomer specific 2-hydroxyacid Amino Acid No

dehydrogenase Metabolism Repression

NCU01830 4-hydroxyphenylpyruvate Amino Acid No

dioxygenase Metabolism Repression

NCU02126 isovaleryl-CoA dehydrogenase Amino Acid No

Metabolism Repression

NCU01744 glutamate synthase Amino Acid Partial

Metabolism Induction-

NCU03748 saccharopine dehydrogenase Amino Acid Partial

Metabolism Induction

NCU06625 cysteine dioxygenase Amino Acid Partial

Metabolism Repression

NCU04130 acylase ACY 1 Amino Acid WT

Metabolism Induction

NCUlOllO 3-hydroxyisobutyrate Amino Acid WT

dehydrogenase Metabolism Induction

NCU03861 glutaminase A Amino Acid WT

Metabolism Repression Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU07623 2,2-dialkylglycine decarboxylase Amino Acid WT

Metabolism Repression

NCU01427 geranylgeranyl pyrophosphate Anabolism Carotenoid WT

synthetase Synthesis Repression

NCU03651 NADP-dependent malic enzyme Anabolism Fatty Acid

Synthesis No Induction

NCU02579 FAS1 domain-containing protein Anabolism Fatty Acid No

Synthesis Repression

NCU07307 fatty acid synthase beta subunit Anabolism Fatty Acid Partial dehydratase Synthesis Repression

NCU07308 fatty acid synthase alpha subunit Anabolism Fatty Acid Partial reductase Synthesis Repression

NCU05858 fatty acid oxygenase Anabolism Fatty Acid WT

Synthesis Repression

NCU01013 delta-aminolevulinic acid Anabolism Heme Anabolism

dehydratase No Induction

NCU06189 5-aminolevulinate synthase Anabolism Heme Synthesis No Induction

NCU05165 pyridoxamine phosphate oxidase Anabolism Vitamin

Metabolism No Induction

NCU04865 polyketide synthase 3 Anabolism Partial

Repression

NCU05011 polyketide synthase 2 Anabolism WT

Induction

NCU00762 endoglucanase 3 Carbon Cellulases

Metabolism No Induction

NCU00836 hypothetical protein Carbon Cellulases

Metabolism No Induction

NCU01050 endoglucanase II Carbon Cellulases

Metabolism No Induction

NCU02240 endoglucanase II Carbon Cellulases

Metabolism No Induction

NCU02344 fungal cellulose binding domain- Carbon Cellulases

containing Metabolism No Induction

NCU02916 endoglucanase II Carbon Cellulases

Metabolism No Induction

NCU03328 endoglucanase II Carbon Cellulases

Metabolism No Induction

NCU04854 endoglucanase EG-1 Carbon Cellulases

Metabolism No Induction

NCU05057 endoglucanase EG-1 Carbon Cellulases

Metabolism No Induction

NCU05121 endoglucanase V Carbon Cellulases

Metabolism No Induction

NCU07190 exoglucanase 3 Carbon Cellulases

Metabolism No Induction

NCU07340 exoglucanase 1 Carbon Cellulases

Metabolism No Induction

NCU07760 endoglucanase IV Carbon Cellulases

Metabolism No Induction

NCU07898 endoglucanase IV Carbon Cellulases

Metabolism No Induction

NCU08760 endoglucanase II Carbon Cellulases No Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Metabolism

NCU09680 exoglucanase 2 Carbon Cellulases

Metabolism No Induction

NCU03322 GDSL family lipase Carbon Fatty

Metabolism Acid/Isoprenoid WT

Metabolism Induction

NCU07362 L-lactate ferricytochrome c Carbon Fermentation Partial oxidoreductase Metabolism Repression

NCU03813 formate dehydrogenase Carbon Fermentation WT

Metabolism Induction

NCU04539 L-Iactate dehydrogenase Carbon Fermentation WT

Metabolism Repression

NCU08687 galactokinase Carbon Galactose

Metabolism Utilization No Induction

NCU05133 related to UDP-glucose 4-epimerase Carbon Galactose WT

Metabolism Utilization Induction

NCU09705 GAL10 Carbon Galactose WT

Metabolism Utilization Induction

NCU07277 anchored cell wall protein 8 Carbon Glycogen/Starch WT

Metabolism Utilization Induction

NCU04797 fructose-l,6-bisphosphatase Carbon Glycolysis No

Metabolism Repression

NCU00575 glucokinase Carbon Glycolysis Partial

Metabolism Induction

NCU04401 fructose-bisphosphate aldolase Carbon Glycolysis WT

Metabolism Induction

NCU02855 endo-l,4-beta-xylanase A Carbon Hemicellulases

Metabolism No Induction

NCU05924 endo-l,4-beta-xyIanase Carbon Hemicellulases

Metabolism No Induction

NCU05955 Cel74a Carbon Hemicellulases

Metabolism No Induction

NCU07326 hypothetical protein Carbon Hemicellulases

Metabolism No Induction

NCU09775 alpha-N-arabinofuranosidase Carbon Hemicellulases

Metabolism No Induction

NCU04997 xylanase Carbon Hemicellulases Partial

Metabolism Induction

NCU01900 xylosidase/arabinosidase Carbon Hemicellulases WT

Metabolism Induction

NCU02343 alpha-L-arabinofuranosidase 2 Carbon Hemicellulases WT

Metabolism Induction

NCU07225 endo-l,4-beta-xylanase 2 Carbon Hemicellulases WT

Metabolism Induction

NCU08087 hypothetical protein Carbon Hemicellulases WT

Metabolism Induction

NCU08189 endo-l,4-beta-xylanase Carbon Hemicellulases WT

Metabolism Induction

NCU09652 beta-xylosidase Carbon Hemicellulases WT

Metabolism Induction

NCU06881 succinyl-CoA:3-ketoacid-coenzyme Carbon Ketone No

A transferase Metabolism Metabolism Repression Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU01853 choline dehydrogenase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU02287 acyl-CoA dehydrogenase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU02894 flavin-binding monooxygenase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU07263 carnitine/acyl carnitine carrier Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU08924 acyl-CoA dehydrogenase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU09692 phosphatidic acid phosphatase beta Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU04796 3-ketoacyl-CoA thiolase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU09732 acetyl-CoA acetyltransferase Carbon Lipid/Isoprenoid No

Metabolism Metabolism Repression

NCU07719 isopentenyl-diphosphate delta- Carbon Lipid/Isoprenoid Partial isomerase Metabolism Metabolism Repression

NCU12093 N-acyl-phosphatidylethanolamine- Carbon Lipid/Isoprenoid Partial hydrolyzing Metabolism Metabolism Repression

NCU05818 phosphatidyl synthase Carbon Lipid/Isoprenoid WT

Metabolism Metabolism Repression

NCU04078 NAD-dependent methanol Carbon Methanol No

dehydrogenase Metabolism Oxidation Repression

NCU07617 Acrl Carbon Mitochondrial WT

Metabolism Carrier Repression

NCU08398 aldose 1-epinierase Carbon Monnosaccharide

Metabolism Metabolism No Induction

NCU10683 NRS/ER Carbon Monnosaccharide

Metabolism Metabolism No Induction

NCU10063 sugar isomerase Carbon Monnosaccharide Partial

Metabolism Metabolism Repression

NCU04933 nucleoside-diphosphate-sugar Carbon Monnosaccharide WT

epimerase Metabolism Metabolism Induction

NCU00890 beta-mannosidase Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU04623 beta-galactosidase Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU04952 beta-D-glucoside glucohydrolase Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU05956 beta-galactosidase Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU07487 periplasmic beta-glucosidase Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU08755 beta-glucosidase 1 Carbon Oligosaccharide

Metabolism Degredation No Induction

NCU00130 beta-glucosidase Carbon Oligosaccharide Partial

Metabolism Degredation Induction

NCU00709 beta-xylosidase Carbon Oligosaccharide WT

Metabolism Degredation Induction

NCU04168 hypothetical protein Carbon Oligosaccharide WT

Metabolism Degredation Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU09904 glucan 1,3-beta-glucosidase Carbon Oligosaccharide WT

Metabolism Degredation Induction

NCU09923 beta-xylosidase Carbon Oligosaccharide WT

Metabolism Degredation Induction

NCU03098 glycosyl hydrolase Carbon Oligosaccharide WT

Metabolism Degredation Repression

NCU09028 class I alpha-mannosidase Carbon Oligosaccharide WT

Metabolism Degredation Repression

NCU09281 alpha-glucosidase Carbon Oligosaccharide WT

Metabolism Degredation Repression

NCU10107 ribose 5-phosphate isomerase Carbon Pentose

Metabolism Phosphate WT

Pathway Induction

NCU00206 cellobiose dehydrogenase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU00710 acetyl xylan esterase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU01059 glycosyl hydrolase family 47 protein Carbon Polysaccharide

Metabolism Degradation No Induction

NCU03181 acetylxylan esterase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU04494 acetyl xylan esterase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU05598 rhamnogalacturonase B Carbon Polysaccharide

Metabolism Degradation No Induction

NCU05751 cellulose-binding protein Carbon Polysaccharide

Metabolism Degradation No Induction

NCU08176 pectate lyase A Carbon Polysaccharide

Metabolism Degradation No Induction

NCU08746 starch binding domain-containing Carbon Polysaccharide

protein Metabolism Degradation No Induction

NCU08785 fungal cellulose binding domain- Carbon Polysaccharide

containing Metabolism Degradation No Induction

NCU09445 Cip2 Carbon Polysaccharide

Metabolism Degradation No Induction

NCU09491 feruloyl esterase B Carbon Polysaccharide

Metabolism Degradation No Induction

NCU09582 chitin deacetylase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU09764 hypothetical protein Carbon Polysaccharide

Metabolism Degradation No Induction

NCU09774 celluiase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU09976 rhamnogalacturonan acetylesterase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU10045 pectinesterase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU11068 endo-beta-l,4-mannanase Carbon Polysaccharide

Metabolism Degradation No Induction

NCU11198 arabinogalactan endo-l,4-beta- Carbon Polysaccharide

galactosidase Metabolism Degradation No Induction

NCU02904 alpha/beta hydrolase fold protein Carbon Polysaccharide Partial Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Metabolism Degradation Induction

NCU04870 acetyl xylan esterase Carbon Polysaccharide Partial

Metabolism Degradation Induction

NCU05159 acetylxylan esterase Carbon Polysaccharide Partial

Metabolism Degradation Induction

NCU09518 glucooligosaccharide oxidase Carbon Polysaccharide Partial

Metabolism Degradation Induction

NCU09664 acetylxylan esterase Carbon Polysaccharide Partial

Metabolism Degradation Induction

NCU09924 BNR/Asp-box repeat protein Carbon Polysaccharide Partial

Metabolism Degradation Induction

NCU03158 alpha/beta hydrolase Carbon Polysaccharide Partial

Metabolism Degradation Repression

NCU07067 mannosyl-oligosaccharide alpha- Carbon Polysaccharide Partial

1,2-mannosidase Metabolism Degradation Repression

NCU01353 mixed-linked glucanase Carbon Polysaccharide WT

Metabolism Degradation Induction

NCU07269 alpha-l,2-mannosidase Carbon Polysaccharide WT

Metabolism Degradation Repression

NCU06023 catabolic 3-dehydroquinase Carbon Quinnic Acid Partial

Metabolism Utilization Repression

NCU06025 shikimate/quinate 5-dehydrogenase Carbon Quinnic Acid WT

Metabolism Utilization Repression

NCU00761 triacylglycerol lipase Carbon Secreted

Metabolism Lipases/Esterases No Induction

NCU06650 secretory phospholipase A2 Carbon Secreted

Metabolism Lipases/Esterases No Induction

NCU09416 cellulose-binding GDSL Carbon Secreted Partial lipase/acylhydrolase Metabolism Lipases/Esterases Induction

NCU00292 cholinesterase Carbon Secreted WT

Metabolism Lipases/Esterases Induction

NCU03903 lipase/esterase Carbon Secreted WT

Metabolism Lipases/Esterases Induction

NCU04475 lipase B Carbon Secreted WT

Metabolism Lipases/Esterases Induction

NCU06364 GDSL lipase/acylhydrolase Carbon Secreted WT

Metabolism Lipases/Esterases Induction

NCU09575 sterol esterase Carbon Secreted WT

Metabolism Lipases/Esterases Repression

NCU04230 isocitrate lyase Carbon TCA No

Metabolism Repression

NCU02366 aconitase Carbon TCA WT

Metabolism Repression

NCU04280 aconitate hydratase Carbon TCA WT

Metabolism Repression

NCU04385 3-isopropylmalate dehydratase Carbon TCA WT

Metabolism Repression

NCU02969 alkaline ceramidase Carbon Transcription WT

Metabolism Factors Repression

NCU08164 retinol dehydrogenase 13 Carbon Vitamin Partial

Metabolism Metabolism Induction

NCU00891 xylitol dehydrogenase Carbon Xylose Utilization WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Metabolism Induction

NCU08384 xylose reductase Carbon Xylose Utilization WT

Metabolism Induction

NCU08272 cytochrome b2 Carbon No

Metabolism Repression

NCU07619 FAD binding domain-containing Carbon Partial protein Metabolism Repression

NCU05304 nuclear segregation protein Cell Cycle No Induction

NCU01510 meiotically up-regulated 190 Cell Cycle No

protein Repression

NCU05768 mating response protein POI2 Cell Cycle Partial

Repression

NCU07154 yippee family protein Cell Cycle Partial

Repression

NCU01998 septin Cell Cycle WT

Induction

NCU08457 rodlet protein Cellular Ascospore WT

Components Repression

NCU06386 dolichyl-phosphate beta- Cellular Cell Wall

glucosyltransferase Components Synthesis or

Modifcation No Induction

NCU09425 NdvB protein Cellular Cell Wall

Components Synthesis or Partial

Modifcation Induction

NCU02478 alpha-l,3-glucan synthase Ags2 Cellular Cell Wall

Components Synthesis or WT

Modifcation Induction

NCU09175 GPI-anchored cell wall beta-1,3- Cellular Cell Wall

endoglucanase Components Synthesis or WT

Modifcation Induction

NCU01689 mitochondrial DNA replication Cellular Mitochondrial

protein YHM2 Components Reproduction No Induction

NCU11721 mitochondrial inner membrane Cellular Mitochondrial

protease subunit 2 Components Reproduction No Induction

NCU02396 mitochondrial FAD-Iinked Cellular Mitochondrial No

sulfhydryl oxidase Components Reproduction Repression

NCU07481 morphogenesis protein Cellular Morphology WT

Components Induction

NCU03137 nuclear elongation and deformation Classification

protein 1 Unclear No Induction

NCU02500 clock-controlled pheromone CCG-4 Clock Controlled No Induction

NCU00565 lipoic acid synthetase Cofaciors No

Repression

NCU02705 FIFO ATP synthase assembly Electron No

protein AtplO transport chain Repression

NCU05225 mitochondrial NADH Electron Partial dehydrogenase transport chain Repression

NCU08326 mitochondrial carrier protein Fermentation No

LEU5 Repression

NCU00326 calcium homeostasis protein Ion Binding

Regucalcin No Induction

NCU08691 EF-hand calcium-binding domain- Ion Binding WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster containing Repression

NCU09043 caleosin domain-containing protein Lipid Associated Partial

Repression

NCU07432 tetraspanin Membrane Partial

Associated Induction

NCU05841 UMTA Methyl Partial transferase Induction

NCU02361 formamidase Nitrogen and

Sulfur No

Metabolism Repression

NCU10051 flavohemoglobin Nitrogen Partial

Metabolism Induction

NCU04720 nitrite reductase Nitrogen WT

Metabolism Induction

NCU04698 spermine/spermidine synthase Nucleotide Partial

Binding Induction

NCU00177 phosphoribosylformylglycinamidine Nucleotide

cyclo-ligase Metabolism No Induction

NCU01786 ribose-phosphate Nucleotide

pyrophosphokinase II Metabolism No Induction

NCU03117 inosine-5'-monophosphate Nucleotide

dehydrogenase IMD2 Metabolism No Induction

NCU05254 ribose-phosphate Nucleotide

pyrophosphokinase Metabolism No Induction

NCU03963 5'-methylthioadenosine Nucleotide

phosphorylase Metabolism No Induction

NCU09659 5'-nucleotidase Nucleotide No

Metabolism Repression

NCU03488 orotidine-5'-phosphate Nucleotide Partial decarboxylase Pyr-4 Metabolism Repression

NCU02657 s-adenosylmethionine synthetase Other No Induction

NCU05855 O-methyltransferase Other No Induction

NCU08044 oxidoreductase Other No Induction

NCU09283 acetyltransferase Other No Induction

NCU11243 alcohol dehydrogenase Other No Induction

NCU01378 acetoacetyl-CoA synthase Other No

Repression

NCU01861 short chain Other No

dehydrogenase/reductase family Repression

NCU04583 acetyltransferase Other No

Repression

NCU06616 S-adenosyimethionine-dependent Other No

Repression

NCU07325 conidiation-specific protein con-10 Other No

Repression

NCU08771 acetolactate synthase Other No

Repression

NCU09553 3-hydroxybutyryl CoA Other No

dehydrogenase Repression

NCU10055 opsin-1 Other No

Repression Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU11289 aldo-keto reductase Other No

Repression

NCU08750 isoamyl alcohol oxidase Other Partial

Induction

NCU08752 acetylcholinesterase Other Partial

Induction

NCU03049 flavin-binding monooxygenase Other Partial

Repression

NCU05653 carbonic anhydrase Other Partial

Repression

NCU07133 metallo-beta-lactamase superfamily Other Partial protein Repression

NCU08925 amine oxidase Other Partial

Repression

NCU09865 methylase Other Partial

Repression

NCU11365 aminotransferase Other Partial

Repression

NCU07055 monooxygenase Other WT

Induction

NCU07224 monooxygenase Other WT

Induction

NCU01061 dienelactone hydrolase Other WT

Repression

NCU03566 short chain Other WT

dehydrogenase/reductase Repression

NCU04260 oxidoreductase domain-containing Other WT

protein Repression

NCU05094 short chain Other WT

dehydrogenase/reductase Repression

NCU05986 sucrase/ferredoxin domain- Other WT

containing protein Repression

NCU06153 monooxygenase Other WT

Repression

NCU09674 O-methyltransferase family 3 Other WT

Repression

NCU11241 nuclease domain-containing protein Other WT

Repression

NCU03013 anchored cell wall protein 10 Oxidoreductase Superoxide WT

dismutase Induction

NCU05319 LysM domain-containing protein Peptidoglycan Partial

Binding Induction

NCU04430 leupeptin-inactivating enzyme 1 Protease Protease WT

Activator Induction

NCU02059 endothiapepsin Protease No Induction

NCU00831 extracellular serine Protease No

carboxypeptidase Repression

NCU06055 extracellular alkaline protease Protease Partial

Induction

NCU00263 serin endopeptidase Protease WT

Induction

NCU07200 metalloprotease 1 Protease WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Induction

NCU09992 serine peptidase Protease WT

Induction

NCU09265 calreticulin Protein Synthesis Protein Folding

and Secretion No Induction

NCU00813 disulfide isomerase Protein Synthesis Protein Folding

and Secretion No Induction

NCU02455 FKBP-type peptidyl-prolyl cis-trans Protein Synthesis Protein Folding

isomerase and Secretion No Induction

NCU09223 protein disulfide-isomerase Protein Synthesis Protein Folding

and Secretion No Induction

NCU09485 related to stress protein ORP150 Protein Synthesis Protein Folding

and Secretion No Induction

NCU01648 dolichyl-phosphate-mannose- Protein Synthesis Protein

protein and Secretion Modification No Induction

NCU10497 oligosaccharyl transferase STT3 Protein Synthesis Protein

subunit and Secretion Modification No Induction

NCU00669 oligosaccharyl transferase subunit Protein Synthesis Protein

and Secretion Modification No Induction

NCU02118 palmitoyltransferase PFA4 Protein Synthesis Protein No

and Secretion Modification Repression

NCU10762 UDP-N-acetyl-glucosamine-l-P Protein Synthesis Protein Partial transferase Alg7 and Secretion Modification Induction

NCU00244 glycosyl transferase Protein Synthesis Protein WT

and Secretion Modification Repression

NCU01068 BAR domain-containing protein Protein Synthesis Protein

and Secretion Trafficing No Induction

NCU03319 COPII-coated vesicle protein Protein Synthesis Protein

SurF4/Erv29 and Secretion Trafficing No Induction

NCU08761 vacuolar sorting receptor Protein Synthesis Protein

and Secretion Trafficing No Induction

NCU01279 ER membrane protein Protein Synthesis Protein No

and Secretion Trafficing Repression

NCU03819 COPII coat assembly protein sec-16 Protein Synthesis Protein Partial and Secretion Trafficing Induction

NCU08607 endoplasmic reticulum-Golgi Protein Synthesis Protein Partial intermediate and Secretion Trafficing Induction

NCU09195 vacuolar membrane PQ loop repeat Protein Synthesis Protein Partial protein and Secretion Trafficing Repression

NCU07736 PEP5 Protein Synthesis Protein WT

and Secretion Trafficing Induction

NCU01290 centromere/microtubule-biiiding Protein Synthesis rRNA

protein CBF5 and Secretion Production No Induction

NCU03396 nucleolar protein nop-58 Protein Synthesis rRNA

and Secretion Production No Induction

NCU09521 ribosome biogenesis protein Protein Synthesis rRNA

and Secretion Production No Induction

NCU03897 RNA binding effector protein Protein Synthesis Translation

Sep 160 and Secretion No Induction

NCU07746 F-box domain-containing protein Protein Synthesis Translocation

and Secretion No Induction

NCU08897 protein transporter SEC61 subunit Protein Synthesis Translocation No Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster alpha and Secretion

NCU00169 translocation complex componenet Protein Synthesis Translocation

and Secretion No Induction

NCU02681 translocation protein Protein Synthesis Translocation

and Secretion No Induction

NCU06333 translocation protein SEC62 Protein Synthesis Translocation

and Secretion No Induction

NCU01146 signal sequence receptor alpha Protein Synthesis Translocation Partial chain and Secretion Induction

NCU00931 lysyl-tRNA synthetase Protein Synthesis tRNA Charging

and Secretion No Induction

NCU07008 carotenoid oxygenase 1 Secondary Carotenoid WT

Metabolism Synthesis Repression

NCU03295 4-coumarate-CoA ligase 1 Secondary No

Metabolism Repression

NCU07737 salicylate hydroxylase Secondary WT

Metabolism Induction

NCU08038 CAS1 Signal Adenlyate Partial

Transduction Cyclase Control Induction

NCU02729 transducin family protein Signal

Transduction No Induction

NCU03364 DENN domain-containing protein Signal

Transduction No Induction

NCU03817 FMI1 protein Signal Partial

Transduction Repression

NCU06111 GTPase Ras2p Signal WT

Transduction Repression

NCU08115 DNA mismatch repair protein Stress Response Partial

Msh3 Induction

NCU06931 sulfite oxidase Sulfur No

Metabolism Repression

NCU04077 assimilatory sulfite reductase Sulfur Partial

Metabolism Induction

NCU01862 SWIRM domain-containing protein Transcriptional Chromatin No

FUN19 Regulation Remodeling Repression

NCU02795 histone deacetylase phdl Transcriptional Chromatin WT

Regulation Remodeling Induction

NCU00812 exosome complex exonuclease Transcriptional RNA processing

RRP41 Regulation No Induction

NCU01856 transcriptional activator hacl Transcriptional Transcription

Regulation Factors No Induction

NCU03725 VIB-1 Transcriptional Transcription

Regulation Factors No Induction

NCU06971 transcriptional activator xlnR Transcriptional Transcription

Regulation Factors No Induction

NCU07705 C6 finger domain-containing Transcriptional Transcription

protein Regulation Factors No Induction

NCU08042 fungal specific transcription factor Transcriptional Transcription

Regulation Factors No Induction

NCU03643 cutinase transcription factor 1 beta Transcriptional Transcription No

Regulation Factors Repression

NCU03043 C2H2 finger domain-containing Transcriptional Transcription Partial

Gene Annotation Group 1 Group 2 Cluster

Repression

NCU05585 MFS quinate transporter Transporter Quinate Partial

Repression

NCU06138 quinate permease Transporter Quinate WT

Induction

NCU05591 ABC transporter CDR4 Transporter Trehalose Export WT

Induction

NCU06032 long-chain fatty acid transporter Transporter No Induction

NCU09098 tetracycline transporter Transporter No Induction

NCU10009 ATP-binding cassette transporter Transporter No Induction

NCU00290 ABC transporter Transporter No

Repression

NCU09580 MSF membrane transporter Transporter No

Repression

NCU00803 MFS transporter, variant Transporter Partial

Repression

NCU04374 MFS transporter Transporter Partial

Repression

NCU08425 major facilitator superfamily Transporter Partial transporter MFS 1 Repression

NCU04097 ABC transporter Transporter WT

Induction

NCU05079 MFS peptide transporter Transporter WT

Induction

NCU07546 multidrug resistance protein MDR Transporter WT

Induction

NCU08148 H+/nucleoside cotransporter Transporter WT

Induction

NCU03107 MFS transporter Transporter WT

Repression

NCU00586 non-anchored cell wall protein 6 Unknown Cell No

Wall Repression

NCU00716 non-anchored cell wall protein 5 Unknown Cell No

Wall Repression

NCU00025 integral membrane protein Unknown

Membrane Partial

Proteins Repression

NCU00848 integral membrane protein TmpA Unknown

Membrane WT

Proteins Repression

NCU00449 hypothetical protein Unknown

Secreted No Induction

NCU00849 hypothetical protein Unknown

Secreted No Induction

NCU01058 hypothetical protein Unknown

Secreted No Induction

NCU01076 hypothetical protein Unknown

Secreted No Induction

NCU01196 hypothetical protein Unknown

Secreted No Induction

NCU01978 hypothetical protein Unknown

Secreted No Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU02138 hypothetical protein Unknown

Secreted No Induction

NCU03083 hypothetical protein Unknown

Secreted No Induction

NCU03982 glucose-regulated protein Unknown

Secreted No Induction

NCU04948 hypothetical protein Unknown

Secreted No Induction

NCU05230 hypothetical protein Unknown

Secreted No Induction

NCU05863 hypothetical protein Unknown

Secreted No Induction

NCU05864 hypothetical protein Unknown

Secreted No Induction

NCU06152 hypothetical protein Unknown

Secreted No Induction

NCU06607 hypothetical protein Unknown

Secreted No Induction

NCU08756 hypothetical protein Unknown

Secreted No Induction

NCU08790 hypothetical protein Unknown

Secreted No Induction

NCU09295 hypothetical protein Unknown

Secreted No Induction

NCU09524 hypothetical protein Unknown

Secreted No Induction

NCU11268 hypothetical protein Unknown

Secreted No Induction

NCU11542 hypothetical protein Unknown

Secreted No Induction

NCU11753 hypothetical protein Unknown

Secreted No Induction

NCU00175 hypothetical protein Unknown No

Secreted Repression

NCU00250 hypothetical protein Unknown No

Secreted Repression

NCU00322 hypothetical protein Unknown No

Secreted Repression

NCU00695 hypothetical protein Unknown No

Secreted Repression

NCU07311 hypothetical protein Unknown No

Secreted Repression

NCU08171 anchored cell wall protein 12 Unknown No

Secreted Repression

NCU08521 hypothetical protein Unknown No

Secreted Repression

NCU10507 hypothetical protein Unknown No

Secreted Repression

NCU07143 6-phosphogluconolactonase Unknown Partial

Secreted Induction

NCU07222 hypothetical protein Unknown Partial

Secreted Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU08371 hypothetical protein Unknown Partial

Secreted Induction

NCU09506 hypothetical protein Unknown Partial

Secreted Induction

NCU04106 hypothetical protein Unknown Partial

Secreted Repression

NCU06526 hypothetical protein Unknown Partial

Secreted Repression

NCU09196 hypothetical protein Unknown Partial

Secreted Repression

NCU11466 hypothetical protein Unknown Partial

Secreted Repression

NCU11957 hypothetical protein Unknown Partial

Secreted Repression

NCU00995 hypothetical protein Unknown WT

Secreted Induction

NCU01720 hypothetical protein Unknown WT

Secreted Induction

NCU03293 hypothetical protein Unknown WT

Secreted Induction

NCU04169 hypothetical protein Unknown WT

Secreted Induction

NCU04170 hypothetical protein Unknown WT

Secreted Induction

NCU04467 hypothetical protein Unknown WT

Secreted Induction

NCU04932 hypothetical protein Unknown WT

Secreted Induction

NCU04998 hypothetical protein Unknown WT

Secreted Induction

NCU05134 hypothetical protein Unknown WT

Secreted Induction

NCU05350 hypothetical protein Unknown WT

Secreted Induction

NCU05829 hypothetical protein Unknown WT

Secreted Induction

NCU05852 glucuronan lyase A Unknown WT

Secreted Induction

NCU05908 hypothetical protein Unknown WT

Secreted Induction

NCU06143 hypothetical protein Unknown WT

Secreted Induction

NCU06983 hypothetical protein Unknown WT

Secreted Induction

NCU06991 hypothetical protein Unknown WT

Secreted Induction

NCU08635 hypothetical protein Unknown WT

Secreted Induction

NCU09046 hypothetical protein Unknown WT

Secreted Induction

NCU09172 hypothetical protein Unknown WT

Gene Annotation Group 1 Group 2 Cluster

NCU09424 hypothetical protein Unknown WT

Secreted Induction

NCU09498 hypothetical protein Unknown WT

Secreted Induction

NCU09823 hypothetical protein Unknown WT

Secreted Induction

NCU09848 hypothetical protein Unknown WT

Secreted Induction

NCU10014 hypothetical protein Unknown WT

Secreted Induction

NCU10039 hypothetical protein Unknown WT

Secreted Induction

NCU10687 hypothetical protein Unknown WT

Secreted Induction

NCU00561 hypothetical protein Unknown WT

Secreted Repression

NCU00859 hypothetical protein Unknown WT

Secreted Repression

NCU02042 hypothetical protein Unknown WT

Secreted Repression

NCU02164 hypothetical protein Unknown WT

Secreted Repression

NCU04482 hypothetical protein Unknown WT

Secreted Repression

NCU04486 hypothetical protein Unknown WT

Secreted Repression

NCU05236 hypothetical protein Unknown WT

Secreted Repression

NCU05761 hypothetical protein Unknown WT

Secreted Repression

NCU05763 hypothetical protein Unknown WT

Secreted Repression

NCU06328 hypothetical protein Unknown WT

Secreted Repression

NCU07948 hypothetical protein Unknown WT

Secreted Repression

NCU08140 hypothetical protein Unknown WT

Secreted Repression

NCU08447 hypothetical protein Unknown WT

Secreted Repression

NCU09734 hypothetical protein Unknown WT

Secreted Repression

NCU12011 hypothetical protein Unknown WT

Secreted Repression

NCU00408 hypothetical protein No Induction

NCU00633 hypothetical protein No Induction

NCU00870 hypothetical protein No Induction

NCU00871 hypothetical protein No Induction

NCU00965 hypothetical protein No Induction

NCU01003 hypothetical protein No Induction

NCU01049 hypothetical protein No Induction Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU01077 hypothetical protein No Induction

NCU01148 methyltransferase No Induction

NCU01944 hypothetical protein No Induction

NCU01970 DUF718 domain-containing protein No Induction

NCU01983 hypothetical protein No Induction

NCU02008 hypothetical protein No Induction

NCU02061 hypothetical protein No Induction

NCU02600 DUF1479 domain-containing

protein No Induction

NCU02625 hypothetical protein No Induction

NCU02720 hypothetical protein No Induction

NCU02915 hypothetical protein No Induction

NCU03152 DUF1348 domain-containing

protein No Induction

NCU03329 hypothetical protein No Induction

NCU03433 hypothetical protein No Induction

NCU04127 hypothetical protein No Induction

NCU04522 hypothetical protein No Induction

NCU04830 hypothetical protein No Induction

NCU04905 hypothetical protein No Induction

NCU05056 hypothetical protein No Induction

NCU05170 hypothetical protein No Induction

NCU05569 hypothetical protein No Induction

NCU05574 hypothetical protein, variant No Induction

NCU05846 hypothetical protein No Induction

NCU05848 cytochrome P450 monooxygenase No Induction

NCU05854 hypothetical protein No Induction

NCU06214 hypothetical protein No Induction

NCU06312 hypothetical protein No Induction

NCU06704 hypothetical protein No Induction

NCU07207 hypothetical protein No Induction

NCU07336 hypothetical protein No Induction

NCU07339 hypothetical protein No Induction

NCU07453 hypothetical protein No Induction

NCU07897 hypothetical protein No Induction

NCU07979 hypothetical protein No Induction

NCU08043 hypothetical protein No Induction

NCU08113 hypothetical protein No Induction

NCU08117 hypothetical protein No Induction

NCU08379 hypothetical protein No Induction

NCU08624 hypothetical protein No Induction

NCU08784 hypothetical protein No Induction

NCU09003 hypothetical protein No Induction

NCU09426 hypothetical protein No Induction

NCU09479 hypothetical protein No Induction

NCU09522 hypothetical protein No Induction

NCU09523 hypothetical protein No Induction

NCU09689 hypothetical protein No Induction

NCU10521 hypothetical protein No Induction

NCU11118 hypothetical protein No Induction

- I l l - Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

NCU11278 hypothetical protein No Induction

NCU11327 No Induction

NCU11397 No Induction

NCU11690 hypothetical protein No Induction

NCU11722 No Induction

NCU11862 hypothetical protein No Induction

NCU00247 hypothetical protein No

Repression

NCU01347 hypothetical protein No

Repression

NCU01598 methyltransferase No

Repression

NCU03761 hypothetical protein No

Repression

NCU04635 hypothetical protein No

Repression

NCU04667 hypothetical protein No

Repression

NCU05058 hypothetical protein No

Repression

NCU05128 hypothetical protein No

Repression

NCU06265 hypothetical protein No

Repression

NCU06615 hypothetical protein No

Repression

NCU06895 cytochrome P450 4A5 No

Repression

NCU07233 hypothetical protein No

Repression

NCU07423 hypothetical protein No

Repression

NCU07424 hypothetical protein No

Repression

NCU07895 hypothetical protein No

Repression

NCU08418 tripeptidyl-peptidase No

Repression

NCU08557 hypothetical protein No

Repression

NCU08712 hypothetical protein No

Repression

NCU09060 hypothetical protein No

Repression

NCU09231 DUF1275 domain-containing No

protein Repression

NCU09685 hypothetical protein No

Repression

NCU09958 hypothetical protein No

Repression

NCU10276 hypothetical protein No Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Repression

NCU11697 No

Repression

NCU11944 No

Repression

NCU12051 hypothetical protein No

Repression

NCU12128 No

Repression

NCU12145 hypothetical protein No

Repression

NCU00289 hypothetical protein Partial

Induction

NCU00496 hypothetical protein Partial

Induction

NCU00763 hypothetical protein Partial

Induction

NCU01386 hypothetical protein Partial

Induction

NCU02485 hypothetical protein Partial

Induction

NCU02882 hypothetical protein Partial

Induction

NCU04618 hypothetical protein Partial

Induction

NCU04871 hypothetical protein Partial

Induction

NCU04904 hypothetical protein Partial

Induction

NCU05351 hypothetical protein Partial

Induction

NCU05501 hypothetical protein Partial

Induction

NCU05906 hypothetical protein Partial

Induction

NCU06373 hypothetical protein Partial

Induction

NCU07270 hypothetical protein Partial

Induction

NCU08116 hypothetical protein Partial

Induction CU08397 hypothetical protein Partial

Induction

NCU08748 hypothetical protein Partial

Induction

NCU08867 hypothetical protein Partial

Induction

NCU09176 hypothetical protein Partial

Induction

NCU11769 Partial

Induction

NCU11828 Partial Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Induction

NCU11905 Partial

Induction

NCU00011 hypothetical protein Partial

Repression

NCU00397 hypothetical protein Partial

Repression

NCU00510 hypothetical protein Partial

Repression

NCU00935 hypothetical protein Partial

Repression

NCU01880 hypothetical protein Partial

Repression

NCU02080 hypothetical protein Partial

Repression

NCU02130 hypothetical protein Partial

Repression

NCU02163 hypothetical protein Partial

Repression

NCU02365 hypothetical protein Partial

Repression

NCU03157 hypothetical protein Partial

Repression

NCU03352 hypothetical protein Partial

Repression

NCU03398 hypothetical protein Partial

Repression

NCU03570 hypothetical protein Partial

Repression

NCU04282 hypothetical protein Partial

Repression

NCU04342 hypothetical protein Partial

Repression

NCU04360 hypothetical protein Partial

Repression

NCU04525 hypothetical protein Partial

Repression

NCU04866 hypothetical protein Partial

Repression

NCU05784 hypothetical protein Partial

Repression

NCU05951 hypothetical protein Partial

Repression

NCU05976 hypothetical protein Partial

Repression

NCU06156 hypothetical protein Partial

Repression

NCU06986 DUF221 domain-containing protein Partial

Repression

NCU07126 hypothetical protein Partial

Repression

NCU07593 hypothetical protein Partial Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Repression

NCU07718 hypothetical protein Partial

Repression

NCU08224 hypothetical protein Partial

Repression

NCU08469 hypothetical protein Partial

Repression

NCU08726 hypothetical protein Partial

Repression

NCU09049 hypothetical protein Partial

Repression

NCU09115 cytochrome P450 52 All Partial

Repression

NCU09883 hypothetical protein Partial

Repression

NCU10658 hypothetical protein Partial

Repression

NCU10770 hypothetical protein Partial

Repression

NCU11294 Partial

Repression

NCU00304 hypothetical protein WT

Induction

NCU00798 hypothetical protein WT

Induction

NCU01136 hypothetical protein WT

Induction

NCU01430 hypothetical protein WT

Induction

NCU03791 hypothetical protein WT

Induction

NCU04167 hypothetical protein WT

Induction

NCU04400 hypothetical protein WT

Induction

NCU04557 hypothetical protein WT

Induction

NCU04879 hypothetical protein WT

Induction

NCU04910 hypothetical protein WT

Induction CU04928 hypothetical protein WT

Induction

NCU05068 hypothetical protein WT

Induction

NCU05755 hypothetical protein WT

Induction

NCU05826 hypothetical protein WT

Induction

NCU05832 hypothetical protein WT

Induction

NCU05875 hypothetical protein WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Induction

NCU05909 hypothetical protein WT

Induction

NCU06181 hypothetical protein WT

Induction

NCU06235 hypothetical protein WT

Induction

NCU06387 hypothetical protein WT

Induction

NCU07235 hypothetical protein WT

Induction

NCU07510 hypothetical protein WT

Induction

NCU07572 hypothetical protein WT

Induction

NCU07997 hypothetical protein WT

Induction

NCU08383 hypothetical protein WT

Induction

NCU08491 hypothetical protein WT

Induction

NCU08634 hypothetical protein WT

Induction

NCU09075 hypothetical protein WT

Induction

NCU09415 hypothetical protein WT

Induction

NCU09856 hypothetical protein WT

Induction

NCU09874 hypothetical protein WT

Induction

NCU09906 hypothetical protein WT

Induction

NCU10284 WT

Induction

NCU10697 hypothetical protein WT

Induction

NCU11095 hypothetical protein WT

Induction

NCU11291 hypothetical protein WT

Induction

NCU11689 hypothetical protein WT

Induction

NCU11801 WT

Induction

NCU11932 hypothetical protein WT

Induction

NCU00365 hypothetical protein WT

Repression

NCU00375 hypothetical protein WT

Repression

NCU00755 hypothetical protein WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Repression

NCU01109 hypothetical protein WT

Repression

NCU01292 hypothetical protein WT

Repression

NCU01551 hypothetical protein WT

Repression

NCU01649 hypothetical protein WT

Repression

NCU03011 hypothetical protein WT

Repression

NCU03417 hypothetical protein WT

Repression

NCU04285 hypothetical protein WT

Repression

NCU04843 hypothetical protein WT

Repression

NCU04851 hypothetical protein WT

Repression

NCU04861 hypothetical protein WT

Repression

NCU04862 hypothetical protein WT

Repression

NCU05006 cytochrome P450 WT

Repression

NCU05189 hypothetical protein WT

Repression

NCU05197 hypothetical protein WT

Repression

NCU05477 hypothetical protein WT

Repression

NCU05762 hypothetical protein WT

Repression

NCU05764 hypothetical protein WT

Repression

NCU05766 hypothetical protein WT

Repression

NCU05859 hypothetical protein WT

Repression

NCU05933 hypothetical protein WT

Repression

NCU06334 hypothetical protein WT

Repression

NCU07180 hypothetical protein WT

Repression

NCU07363 hypothetical protein WT

Repression

NCU08037 hypothetical protein WT

Repression

NCU08155 hypothetical protein WT

Repression

NCU08156 hypothetical protein WT Table 1A: Genes under regulation by cellulose in wild type N. crassa clustered by level of induction/repression in clr mutants

Gene Annotation Group 1 Group 2 Cluster

Repression

NCU08170 hypothetical protein WT

Repression

NCU08455 hypothetical protein WT

Repression

NCU08554 peptidyl-prolyl cis-trans isomerase WT

ssp-1 Repression

NCU08622 hypothetical protein WT

Repression

NCU08700 hypothetical protein WT

Repression

NCU08775 hypothetical protein WT

Repression

NCU09272 hypothetical protein WT

Repression

NCU09273 hypothetical protein WT

Repression

NCU09274 hypothetical protein WT

Repression

NCU09335 hypothetical protein WT

Repression

NCU09342 hypothetical protein WT

Repression

NCU09714 hypothetical protein WT

Repression

NCU09782 hypothetical protein WT

Repression

NCU10062 hypothetical protein WT

Repression

NCU10301 hypothetical protein WT

Repression

NCU11565 hypothetical protein WT

Repression

NCU11774 WT

Repression

NCU11881 hypothetical protein WT

Repression

NCU11974 hypothetical protein WT

Repression

NCU11989 hypothetical protein WT

Repression

NCU12012 WT

Repression

NCU12014 hypothetical protein WT

Repression

NCU12015 hypothetical protein WT

Repression

[0342]

Annotation: Broad Institute Annotation Group 1: Author's hand curated annotation categ

Group 2: Author's hand curated annotation/function

Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

Pathway

NCU03861 Secretory 1

Pathway

NCU07623 Other 2

NCU01427 Other 3

NCU03651 Mitochondrion 3

NCU02579 Secretory 1

Pathway

NCU07307 Mitochondrion 4

NCU07308 Secretory 4

Pathway

NCU05858 Other 2

NCU01013 Mitochondrion 4

NCU06189 Mitochondrion 4

NCU05165 Secretory 1 1

Pathway

NCU04865 Other 2

NCU0501 1 Other 3

NCU00762 CBM1, Secretory 2 Avi/Mis Avi/Mis Cellulase

GH5 Pathway

NCU00836 CBM1, Secretory 4 Avi/Mis Cellulase

GH61 Pathway

NCU01050 GH61 Secretory 2 Avi/Mis Avi/Mis Cellulase

Pathway

NCU02240 CBM1, Secretory 2 Avi Avi/Mis Cellulase

GH61 Pathway

NCU02344 GH61 Secretory 4 Avi/Mis Cellulase

Pathway

NCU02916 CBM1, Secretory 3 Avi/Mis Cellulase

GH61 Pathway

NCU03328 GH61 Secretory 1 Avi/Mis Cellulase

Pathway

NCU04854 GH7 Secretory 2 Avi/Mis Cellulase

Pathway

NCU05057 GH7 Secretory 2 Avi/Mis Avi/Mis Cellulase

Pathway

NCU05121 CBM1, Secretory 2 Avi Avi/Mis Cellulase

GH45 Pathway

NCU07190 GH6 Secretory 3 Avi/Mis Avi/Mis Cellulase

Pathway

NCU07340 CBM1, Secretory 2 Avi/Mis Avi/Mis Cellulase

GH7 Pathway

NCU07760 CBM1, Secretory 2 Mis Cellulase

GH61 Pathway

NCU07898 GH61 Secretory 1 Avi/Mis Avi/Mis Cellulase

Pathway

NCU08760 CBM1, Secretory 1 Avi/Mis Avi/Mis Cellulase

GH61 Pathway

NCU09680 CBM1, Secretory 1 Avi/Mis Avi/Mis Cellulase

GH6 Pathway Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU03322 Other 1

NCU07362 Other 2

NCU03813 Other 2

NCU04539 Other 3

NCU08687 Other 2

NCU05133 Other 2

NCU09705 Secretory 3 Avi/Mis

Pathway

NCU07277 Secretory 1 Avi

Pathway

NCU04797 Other 2

NCU00575 Other 1

NCU04401 Other 4 Mis

NCU02855 GH11 Secretory 3 Avi Avi/Mis Hemicellul

Pathway ase

NCU05924 GH10 Secretory 2 Avi/Mis Avi/Mis Hemicellul

Pathway ase

NCU05955 CBMl, Secretory 2 Avi/Mis Avi/Mis Hemicellul

GH74 Pathway ase

NCU07326 GH43 Secretory 1 Avi/Mis Avi/Mis Hemicellul

Pathway ase

NCU09775 GH54 Secretory 1 Mis Mis Hemicellul

Pathway ase

NCU04997 CBMl, Secretory 2 Hemicellul

GH10 Pathway ase

NCU01900 GH43 Other 2 Avi/Mis Hemicellul ase

NCU02343 GH51 Secretory 1 Mis Avi/Mis Hemicellul

Pathway ase

NCU07225 CBMl, Secretory 2 Avi/Mis Avi/Mis Hemicellul

GH11 Pathway ase

NCU08087 GH26 Other 2 Hemicellul ase

NCU08189 GH10 Secretory 1 Avi/Mis Avi/Mis Hemicellul

Pathway ase

NCU09652 GH43 Other 2 Avi/Mis Hemicellul ase

NCU06881 Mitochondrion 2

NCU01853 Other 2

NCU02287 Other 2

NCU02894 Other 3

NCU07263 Other 3 2

NCU08924 Other 2

NCU09692 Mitochondrion 5 6

NCU04796 Other 2

NCU09732 Mitochondrion 1

NCU07 19 Other 2

NCU 12093 Other 2

NCU05818 Other 3 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU04078 Mitochondrion 2

NCU07617 Other 2

NCU08398 Secretory 4 Avi/Mis Avi/Mis

Pathway

NCU10683 Other 2

NCU10063 Other 2

NCU04933 Secretory 5

Pathway

NCU00890 GH2 Other 3 Avi

NCU04623 GH35 Secretory 4

Pathway

NCU04952 GH3 Secretory 2 Avi/Mis Avi

Pathway

NCU05956 GH2 Other 4

NCU07487 GH3 Other 2 Avi/Mis

NCU08755 GH3 Secretory 2 Avi/Mis

Pathway

NCU00130 GH1 Other 4 Avi/Mis

NCU00709 GH3 Secretory 1

Pathway

NCU04168 GH16 Other 2 1

NCU09904 GH16 Other 1 1 Avi

NCU09923 GH3 Secretory 1 Mis Mis

Pathway

NCU03098 GH15 Other 4

NCU09028 GH47 Mitochondrion 3 1

NCU09281 GH31 Secretory 1

Pathway

NCU10107 Other 2

NCU00206 CBMl Secretory 3 Avi/Mis Avi/Mis

Pathway

NCU00710 CBMl, Secretory 2 Mis

CEl Pathway

NCU01059 GH47 Secretory 3 1

Pathway

NCU03181 Secretory 3 Avi/Mis

Pathway

NCU04494 CEl Secretory 1 Mis

Pathway

NCU05598 PL4 Secretory 1 1 Avi/Mis

Pathway

NCU05751 CE3 Secretory 1 Mis Mis

Pathway

NCU08176 PL3 Secretory 2 Avi/Mis

Pathway

NCU08746 CBM20 Secretory 3 Avi/Mis

Pathway

NCU08785 CEl Secretory 1 Mis Avi/Mis

Pathway

NCU09445 CE15 Secretory 1 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

Pathway

NCU09491 CE1 Secretory 2 Avi/Mis Mis

Pathway

NCU09582 CE4 Secretory Avi/Mis

Pathway ¹

NCU09764 CBM1 Secretory Avi/Mis

Pathway ¹

NCU09774 CE1 Secretory Avi

¹

Pathway

NCU09976 CE12 Secretory Mis

Pathway

NCU 10045 CE8 Secretory Avi/Mis

¹

Pathway

NCU 1 1068 Other

NCU1 1 198 GH53 Secretory

Pathway ¹

NCU02904 Secretory

Pathway ¹

NCU04870 CE1 Secretory Mis Mis

¹

Pathway

NCU05159 CBM1, Secretory Mis Avi/Mis

¹

CE5 Pathway

NCU09518 Secretory

Pathway ¹

NCU09664 CE5 Secretory Avi/Mis

¹

Pathway

NCU09924 GH93 Secretory Mis

Pathway ¹

NCU03158 Secretory 1

Pathway ⁴

NCU07067 GH47 Secretory 3

Pathway ¹

NCU01353 GH16 Secretory 3

Pathway

NCU07269 GH92 Secretory 2

Pathway

NCU06023 Other 3

NCU06025 Other 4

NCU00761 Secretory 1

Pathway

NCU06650 Secretory 1

Pathway

NCU09416 CBM1, Secretory 1 Avi

CE16 Pathway

NCU00292 Secretory 2

Pathway

NCU03903 Secretory 2

Pathway

NCU04475 Secretory 1 Mis

Pathway Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU06364 Secretory 2

Pathway

NCU09575 Secretory 3

Pathway

NCU04230 Other 1

NCU02366 Mitochondrion 2

NCU04280 Mitochondrion 2

NCU04385 Other 2

NCU02969 Other 3 6

NCU08164 Other 2

NCU00891 Other 2 Mis

NCU08384 Other 2 Avi/Mis

NCU08272 Other 1

NCU07619 Secretory 2

Pathway

NCU05304 Other 2

NCU01510 Other 1 1

NCU05768 Secretory 1

Pathway

NCU07154 Other 3

NCU01998 Mitochondrion 4

NCU08457 Secretory 1

Pathway

NCU06386 GT2 Secretory 2 1

Pathway

NCU09425 GH94 Other 5

NCU02478 GT5, Secretory 2 13

GH13 Pathway

NCU09175 GH17 Secretory 3 Avi/Mis Avi/Mis

Pathway

NCU01689 Other 3

NCU1 1721 Mitochondrion 5

NCU02396 Other 2

NCU07481 Other 2

NCU03137 Other 2

NCU02500 Secretory 2 Avi

Pathway

NCU00565 Mitochondrion 1

NCU02705 Mitochondrion 1

NCU05225 Mitochondrion 2 1

NCU08326 Other 2

NCU00326 Other 1 Avi/Mis

NCU08691 Other 3

NCU09043 Other 1 1

NCU07432 Secretory 1 4

Pathway

NCU05841 Other 2

NCU02361 Other 1

NCU10051 Other 4 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU04720 Other 3

NCU04698 Other 2 1

NCU00177 Mitochondrion 5

NCU01786 Other 2

NCU03117 Other 2

NCU05254 Other 2

NCU03963 Other 2

NCU09659 Secretory 2

Pathway

NCU03488 Other 3

NCU02657 Other 2

NCU05855 Other 5 Avi

NCU08044 Mitochondrion 2

NCU09283 Other 2

NCU11243 Other 3

NCU01378 Other 4

NCU01861 Mitochondrion 1

NCU04583 Mitochondrion 4

NCU06616 Mitochondrion 2

NCU07325 Other 1 Avi

NCU08771 Other 4

NCU09553 Mitochondrion 2

NCU10055 Other 3 7

NCU11289 Other 3

NCU08750 Secretory 1 Avi/Mis

Pathway

NCU08752 Secretory 1

Pathway

NCU03049 Other 2

NCU05653 Secretory 4

Pathway

NCU07133 Other 3

NCU08925 Other 3

NCU09865 Other 2

NCU11365 Other 2

NCU07055 Secretory 3

Pathway

NCU07224 Secretory 2

Pathway

NCU01061 Other 4

NCU03566 Other 4

NCU04260 Other 3

NCU05094 Other 4 1

NCU05986 Other 3

NCU06153 Other 2 1

NCU09674 Other 1

NCU11241 Other 1

NCU03013 Secretory 3 Mis

Pathway Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU05319 Other 3 Avi/Mis

NCU04430 Secretory 2

Pathway

NCU02059 Secretory 2 Mis

Pathway

NCU00831 Secretory 3

Pathway

NCU06055 Secretory 2

Pathway

NCU00263 Secretory 2 Mis

Pathway

NCU07200 Secretory 1

Pathway

NCU09992 Secretory 3 Mis

Pathway

NCU09265 Secretory 4 1 Mis

Pathway

NCU00813 Secretory 2 Avi/Mis

Pathway

NCU02455 Secretory 1 Mis

Pathway

NCU09223 Secretory 2 Avi/Mis

Pathway

NCU09485 Secretory 1 1 Mis

Pathway

NCU01648 GT39 Other 2 9

NCU 10497 Other 3 13

NCU00669 Secretory 1 1

Pathway

NCU021 18 Secretory 3 4

Pathway

NCU 10762 Secretory 5 1

Pathway

NCU00244 GT8 Other 3

NCU01068 Other 2

NCU03319 Other 5 6 Mis

NCU08761 Secretory 2

Pathway

NCU01279 Secretory 1 1

Pathway

NCU03819 Other 2

NCU08607 Other 4 2 Mis

NCU09195 Other 5 7

NCU07736 Other 2 14

NCU01290 Other 2

NCU03396 Other 2

NCU09521 Other 1

NCU03897 Other 1

NCU07746 Other 1

NCU08897 Secretory 4 8 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

Pathway

NCU00169 Other 3 3

NCU02681 Secretory 1 1

Pathway

NCU06333 Other 2 2 Mis

NCU01 146 Secretory 1 1

Pathway

NCU00931 Other 3

NCU07008 Other 1

NCU03295 Other 2

NCU07737 Other 3 Avi/Mis

NCU08038 Secretory 1 1 Mis

Pathway

NCU02729 Other 5

NCU03364 Mitochondrion 4

NCU03817 Mitochondrion 1

NCU061 1 1 Mitochondrion 5

NCU081 15 Mitochondrion 5

NCU06931 Mitochondrion 2

NCU04077 Other 3

NCU01862 Other 2

NCU02795 Other 2

NCU00812 Mitochondrion 4

NCU01856 Other 1

NCU03725 Other 2

NCU06971 Mitochondrion 5

NCU07705 Other 4 Avi/Mis

NCU08042 Other 3

NCU03643 Other 1

NCU03043 Other 3

NCU05767 Other 3

NCU00316 Other 4 2

NCU00721 Other 2 12

NCU07578 Other 5

NCU04435 Other 1 12

NCU05198 Other 1 11

NCU 10721 Other 4 8

NCU1 1342 Other 2

NCU00821 Secretory 2 1

Pathway

NCU08561 Other 2

NCU09287 Other 1 11

NCU00801 Other 1 12 Avi/Mis

NCU00809 Other 1 12

NCU07668 Other 5

NCU05089 Other 2 12

NCU08152 Secretory 1 12

Pathway

NCU01633 Other 2 12 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU04537 Other 1 12

NCU05853 Other 2 11 Avi/Mis

NCU08114 Other 2 9 Avi/Mis

NCU00023 Mitochondrion 4 6

NCU02009 Other 3 5

NCU07068 Secretory 4 12

Pathway

NCU03305 Other 3 9

NCU08225 Secretory 2 7

Pathway

NCU08147 Other 1 1

NCU06366 Other 3

NCU05585 Other 3 1 Mis

NCU06138 Other 5 11 Avi/Mis

NCU05591 Other 4 13

NCU06032 Secretory 2

Pathway

NCU09098 Secretory 1 12

Pathway

NCU 10009 Other 2 13

NCU00290 Other 2 6 Avi

NCU09580 Other 1

NCU00803 Other 3 12

NCU04374 Other 1 12

NCU08425 Secretory 2 1

Pathway

NCU04097 Other 2

NCU05079 Other 1 11

NCU07546 Other 1 12 Avi

NCU08148 Other 1

NCU03107 Other 3 1

NCU00586 Secretory 1 4

Pathway

NCU00716 Secretory 1

Pathway

NCU00025 Secretory 1 9

Pathway

NCU00848 Other 1 6

NCU00449 Secretory 1

Pathway

NCU00849 Secretory 3

Pathway

NCU01058 Secretory 4

Pathway

NCU01076 Secretory 1 1 Avi

Pathway

NCU01196 Secretory 1

Pathway

NCU01978 Secretory 1 8 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains a)., MA Annotatio e n

Pathway

NCU02138 Secretory 1 1

Pathway

NCU03083 Secretory 2

Pathway

NCU03982 Secretory 1 1 Avi/Mis

Pathway

NCU04948 Secretory 1

Pathway

NCU05230 Secretory 1 4

Pathway

NCU05863 Secretory 5

Pathway

NCU05864 Secretory 2 Avi/Mis

Pathway

NCU06152 Secretory 2 1

Pathway

NCU06607 Secretory 2 Avi

Pathway

NCU08756 Secretory 2

Pathway

NCU08790 Secretory 1

Pathway

NCU09295 Secretory 1 1

Pathway

NCU09524 Secretory 5 Avi/Mis

Pathway

NCU1 1268 Secretory 4

Pathway

NCU1 1542 Secretory 1

Pathway

NCU 1 1753 Secretory 2

Pathway

NCU00175 Secretory 3

Pathway

NCU00250 Secretory 1 2

Pathway

NCU00322 Secretory 2

Pathway

NCU00695 Secretory 3 Avi

Pathway

NCU0731 1 Secretory 1 4

Pathway

NCU08171 Secretory 2

Pathway

NCU08521 Secretory 1 2

Pathway

NCU 10507 Secretory 1

Pathway

NCU07143 Secretory 1 Avi/Mis Avi/Mis Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

Pathway

NCU07222 Secretory 1

Pathway

NCU08371 Secretory 2 1 Mis

Pathway

NCU09506 Secretory 1

Pathway

NCU04106 Secretory 5 6

Pathway

NCU06526 Secretory 3 1

Pathway

NCU09196 Secretory 2

Pathway

NCU11466 Secretory 4

Pathway

NCU1 1957 Secretory 5

Pathway

NCU00995 Secretory 4

Pathway

NCU01720 Secretory 2

Pathway

NCU03293 Secretory 1

Pathway

NCU04169 Secretory 1 Avi

Pathway

NCU04170 Secretory 2

Pathway

NCU04467 Secretory 1

Pathway

NCU04932 Secretory 2

Pathway

NCU04998 Secretory 1

Pathway

NCU05134 Secretory 1 Avi Avi/Mis

Pathway

NCU05350 Secretory 1 4

Pathway

NCU05829 Secretory 3 7

Pathway

NCU05852 Secretory 1

Pathway

NCU05908 Secretory 2

Pathway

NCU06143 Secretory 2 Avi/Mis

Pathway

NCU06983 Secretory 2

Pathway

NCU06991 Secretory 1 3

Pathway

NCU08635 Secretory 4 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

Pathway

NCU09046 Secretory 1

Pathway

NCU09172 Secretory 5 12

Pathway

NCU09424 Secretory 1

Pathway

NCU09498 Secretory 3 Avi

Pathway

NCU09823 Secretory 1 7

Pathway

NCU09848 Secretory 2

Pathway

NCU10014 Secretory 2 Avi/Mis

Pathway

NCU10039 Secretory 3 1

Pathway

NCU 10687 Secretory 2

Pathway

NCU00561 Secretory 2 1

Pathway

NCU00859 Secretory 1

Pathway

NCU02042 Secretory 1 1

Pathway

NCU02164 Secretory 1 1

Pathway

NCU04482 Secretory 1 Avi

Pathway

NCU04486 Secretory 1 3

Pathway

NCU05236 Secretory 5 1

Pathway

NCU05761 Secretory 5

Pathway

NCU05763 Secretory 1 1

Pathway

NCU06328 Secretory 1 6

Pathway

NCU07948 Secretory 1

Pathway

NCU08140 Secretory 2

Pathway

NCU08447 Secretory 1 7

Pathway

NCU09734 Secretory 2 1

Pathway

NCU1201 1 Secretory 1

Pathway

NCU00408 Other 1 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU00633 Other 2

NCU00870 Other 4 Avi/Mis

NCU00871 Other 2

NCU00965 Other 2 2

NCU01003 Other 3

NCU01049 Other 3

NCU01077 Other 1

NCU01 148 Other 2

NCU01944 Other 4

NCU01970 Other 3 Avi/Mis

NCU01983 Mitochondrion 2

NCU02008 Mitochondrion 3

NCU02061 Mitochondrion 2

NCU02600 Mitochondrion 4

NCU02625 Mitochondrion 5 1

NCU02720 Other 4

NCU02915 Other 3

NCU03152 Other 2

NCU03329 Other 2

NCU03433 Other 3

NCU04127 Other 1 1

NCU04522 Other 3

NCU04830 Other 2

NCU04905 Other 1 Avi/Mis

NCU05056 Other 2

NCU05170 Other 2 1

NCU05569 Other 4

NCU05574 Other 3

NCU05846 Mitochondrion 5 Avi/Mis

NCU05848 Secretory 2 1

Pathway

NCU05854 Other 4 7

NCU06214 Other 4

NCU06312 Other 3 7

NCU06704 Other 5 1

NCU07207 Other 2

NCU07336 Other 4

NCU07339 Other 4 1

NCU07453 Other 2

NCU07897 Other 1

NCU07979 Other 4

NCU08043 Other 3

NCU081 13 Other 2

NCU081 17 Other 4

NCU08379 Mitochondrion 4 1

NCU08624 Other 3 6

NCU08784 Other 3

NCU09003 Other 5

NCU09426 Other 4 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU09479 Other 3 1

NCU09522 Other 5

NCU09523 Other 4

NCU09689 Other 1 Avi/Mis

NCU10521 Other 5

NCU1 1 1 18 Mitochondrion 3

NCU 1 1278 Other 3

NCU 1 1327

NCU1 1397

NCU 1 1690 Other 3

NCU1 1722

NCU 1 1862 Other 1

NCU00247 Mitochondrion 5 1

NCU01347 Mitochondrion 5

NCU01598 Other 2

NCU03761 Other 3

NCU04635 Other 1 Mis

NCU04667 Other 2

NCU05058 Other 1

NCU05128 Other 3

NCU06265 Other 2

NCU06615 Mitochondrion 3 Avi

NCU06895 Secretory 1 1

Pathway

NCU07233 Other 4 1

NCU07423 Mitochondrion 1

NCU07424 Other 5

NCU07895 Other 5

NCU08418 Mitochondrion 5

NCU08557 Other 1

NCU08712 Mitochondrion 2

NCU09060 Other 2

NCU09231 Other 3 4

NCU09685 Mitochondrion 5 1

NCU09958 Other 4

NCU 10276 Other 2 11

NCU1 1697

NCU 1 1944

NCU12051 Mitochondrion 4

NCU12128

NCU12145 Other 2

NCU00289 Other 2

NCU00496 Other 3

NCU00763 Other 2

NCU01386 Other 1

NCU02485 Other 2 Mis

NCU02882 Other 3

NCU04618 Other 2

NCU04871 Mitochondrion 5 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU04904 Other 3

NCU05351 Mitochondrion 4

NCU05501 Other 2 Mis

NCU05906 Other 2

NCU06373 Mitochondrion 5

NCU07270 Other 2

NCU081 16 Other 5

NCU08397 Other 1 11 Avi/Mis

NCU08748 Other 1 11

NCU08867 Other 5 1

NCU09176 Other 3

NCU 11769

NCU1 1828

NCU11905

NCU0001 1 Mitochondrion 3

NCU00397 Other 4

NCU00510 Mitochondrion 4

NCU00935 Other 1

NCU01880 Other 3

NCU02080 Mitochondrion 4

NCU02130 Other 4

NCU02163 Other 4

NCU02365 Other 2

NCU03157 Mitochondrion 5

NCU03352 Other 1

NCU03398 Other 3

NCU03570 Mitochondrion 5

NCU04282 Other 2

NCU04342 Other 2

NCU04360 Other 2

NCU04525 Other 2

NCU04866 Other 4

NCU05784 Other 1

NCU05951 Other 4 1

NCU05976 Other 4

NCU06156 Other 1

NCU06986 Other 4 11

NCU07126 Other 1

NCU07593 Other 5

NCU07718 Other 2

NCU08224 Mitochondrion 4 Mis

NCU08469 Other 3

NCU08726 Other 2

NCU09049 Mitochondrion 5 4

NCU091 15 Other 3 1

NCU09883 Mitochondrion 5 7 Avi

NCU10658 Other 2

NCU10770 Other 2 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU1 1294

NCU00304 Mitochondrion 3 Mis

NCU00798 Mitochondrion 5 Avi Avi

NCU01 136 Other 5

NCU01430 Other 2 Mis

NCU03791 Mitochondrion 2

NCU04167 Other 2 7

NCU04400 Other 5 Mis

NCU04557 Mitochondrion 2

NCU04879 Other 5

NCU04910 Other 3 Avi/Mis

NCU04928 Other 2

NCU05068 Other 3 7 Avi

NCU05755 Other 2 Mis

NCU05826 Other 1 1

NCU05832 Mitochondrion 5

NCU05875 Other 2

NCU05909 Mitochondrion 4

NCU06181 Mitochondrion 4

NCU06235 Other 2

NCU06387 Other 4 Mis

NCU07235 Other 3

NCU07510 Other 2

NCU07572 Other 1

NCU07997 Other 2 Avi/Mis

NCU08383 Mitochondrion 5

NCU08491 Other 5

NCU08634 Mitochondrion 4

NCU09075 Other 1

NCU09415 Other 2

NCU09856 Other 2

NCU09874 Other 1 13

NCU09906 GTNC Other 3 7 Avi

NCU10284

NCU 10697 Mitochondrion 2

NCU 1 1095 Other 2

NCU1 1291 Other 4

NCU1 1689 Other 4

NCU 1 1801

NCU11932 Mitochondrion 1

NCU00365 Other 4

NCU00375 Other 5

NCU00755 Mitochondrion 4 11

NCU01109 Mitochondrion 5

NCU01292 Other 4

NCU01551 Mitochondrion 5

NCU01649 Other 2

NCU0301 1 Mitochondrion 3 Table IB: Annotation information for genes in Table 1A

Gene CAZy Signal P Signal P TM LCMS Tian et Tian et al.

Confidenc Domains al., MA Annotatio e n

NCU03417 Other 3

NCU04285 Other 4

NCU04843 Other 3

NCU04851 Other 2

NCU04861 Other 4

NCU04862 Other 2

NCU05006 Other 4

NCU05189 Other 2 5

NCU05197 Other 2

NCU05477 Other 4

NCU05762 Other 5

NCU05764 Other 1

NCU05766 Other 3

NCU05859 Other 3

NCU05933 Other 2

NCU06334 Other 3

NCU07180 Other 2

NCU07363 Other 3 1

NCU08037 Other 2

NCU08155 Mitochondrion 1

NCU08156 Other 2

NCU08170 Other 5

NCU08455 Mitochondrion 5

NCU08554 Other 1

NCU08622 Other 5

NCU08700 Mitochondrion 4

NCU08775 Mitochondrion 4 1

NCU09272 Other 2

NCU09273 Mitochondrion 1

NCU09274 Other 3

NCU09335 Other 2

NCU09342 Other 2

NCU09714 Other 1

NCU09782 Other 3 4

NCU10062 Other 5

NCU10301 Other 3 1

NCU11565 Other 1

NCU 1 1774

NCU1 1881 Other 5

NCU1 1974 Other 2

NCU1 1989 Other 2

NCU12012

NCU12014 Other 4

NCU12015 Other 4

[0343] [0344] CAZy: Predicted Domains from the Carbohydrate Active Enzymes database

(Cantarelet al., (2009) Nucleic Acids Res 37:D233-238 [PMID: 18838391 ]).

[0345] SignalP: Predicted target location from Signal P (Bendtsen et al., J. Mol. Biol., 340:783-795, 2004).

[0346] TM Domains: Predicted Transmembrane Domains

[0347] LCMS: Condition under which a gene product was detected in the culture supernatant by Tian et al, Proc Natl Acad Sci 2009.

[0348] Tian et al, MA: Condition under which Tian et al, Proc Natl Acad Sci 2009 detected transcriptional changes by microarray.

[0349] Tian et al. Annotation: Classification as cellulase or hemicellulase by Tian et al, Proc Natl Acad Sci 2009.

Table 1C: FPKMs identified from profiling data in different mutant

strains and growth conditions

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU00554 2050 87 313 116 99 551

NCU00944 283 229 422 161 213 252

NCU01195 15484 1792 3179 982 887 16026

NCU02785 1091 62 146 79 78 300

NCU02954 1862 154 250 131 122 1000

NCU03131 149 68 236 97 78 184

NCU04216 1239 8 26 5 5 236

NCU04298 9 105 218 119 140 785

NCU04837 2537 170 336 157 181 694

NCU05548 2782 111 581 63 73 563

NCU07413 35 13 106 19 18 95

NCU 10283 1142 121 522 177 154 410

NCU00461 91 4049 1099 4925 2071 255

NCU00591 22 1360 458 2184 1250 61

NCU00680 555 1138 327 1280 888 387

NCU01402 1 203 58 744 687 5

NCU02127 10 662 177 1585 794 44

NCU02704 14 349 104 1118 533 15

NCU02727 290 220 141 435 185 183

NCU02936 103 451 151 1083 527 40

NCU03076 322 746 311 1571 904 194

NCU03415 43 17491 4745 15467 11754 2948

NCU03648 2 1496 667 2234 1969 221

NCU03913 35 349 146 1194 580 40

NCU05499 11 809 306 2076 1535 28

Table 1C: FPKMs identified from profiling data in different mutant strains and growth conditions

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU03813 14 589 1740 1581 1072 2822

NCU04539 2 57 25 22 30 17

NCU08687 110 283 1342 431 354 1147

NCU05133 957 320 1831 1241 1520 1247

NCU09705 44 266 4525 2740 3468 11549

NCU07277 682 1273 3013 3216 3317 2258

NCU04797 30 877 258 1646 542 167

NCU00575 2198 677 2103 1123 1057 1812

NCU04401 8 66 3833 1630 2316 7742

NCU02855 5 11 4659 63 36 385

NCU05924 5 5 19425 14 4 2339

NCU05955 14 100 4569 88 102 64

NCU07326 18 55 10461 63 43 110

NCU09775 0.01 3 99 10 6 302

NCU04997 5 3 116 16 8 20

NCU01900 28 210 11254 6388 7907 14560

NCU02343 41 458 9436 14639 15125 23514

NCU07225 35 111 104230 7967 5971 24494

NCU08087 72 4 35 83 63 129

NCU08189 27 208 42393 39471 48947 50079

NCU09652 24 340 4032 3208 3390 5729

NCU06881 249 259 169 421 251 125

NCU01853 0.01 123 50 131 131 25

NCU02287 134 703 406 1073 886 94

NCU02894 107 191 83 140 126 113

NCU07263 166 197 68 278 129 24

NCU08924 132 2324 770 1742 1287 205

NCU09692 24 383 124 330 267 71

NCU04796 166 1919 704 1594 1469 135

NCU09732 112 772 430 1876 740 222

NCU07719 865 941 441 572 562 616

NCU 12093 14 279 74 156 136 23

NCU05818 2 29 10 5 5 4

NCU04078 15 360 117 643 316 48

NCU07617 9 196 37 41 49 7

NCU08398 2 52 5368 66 25 84

NCU10683 12 37 99 36 32 41

NCU 10063 45 256 68 112 107 150

NCU04933 35 27 155 249 248 100

NCU00890 18 63 1613 139 58 536

NCU04623 3 40 128 38 69 161

NCU04952 1 62 777 22 32 17

NCU05956 5 398 935 379 446 134

NCU07487 2 6 988 6 13 5

NCU08755 6 468 8742 467 3396 2802

NCU00130 30 159 39866 759 2765 1736

NCU00709 2 6 192 300 311 2803

NCU04168 4 18 166 407 302 40 Table 1C: FPKMs identified from profiling data in different mutant strains and growth conditions

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU09904 13 16 76 111 98 198

NCU09923 1 12 222 92 89 2701

NCU03098 1 360 56 75 107 17

NCU09028 0.01 26 8 7 9 6

NCU09281 16 704 133 171 180 23

NCU10107 0.01 165 2709 1434 2108 3537

NCU00206 12 24 12252 18 23 16

NCU00710 11 11 921 43 44 174

NCU01059 37 42 253 26 91 39

NCU03181 5 32 2023 39 21 49

NCU04494 87 74 529 65 75 143

NCU05598 39 26 313 39 36 144

NCU05751 4 10 1138 5 9 1422

NCU08176 6 18 1547 35 24 268

NCU08746 3 44 289 22 55 639

NCU08785 1 26 5257 3 3 126

NCU09445 1 0.01 28 0.01 2 5

NCU09491 0.01 19 508 21 15 172

NCU09582 9 17 2971 54 60 759

NCU09764 28 9 2438 14 15 29

NCU09774 0.01 0.01 18 0.01 0.01 0.01

NCU09976 3 4 77 3 6 20

NCU 10045 18 71 1641 213 206 1324

NCU11068 6 71 12618 670 6 1972

NCU11198 36 40 742 47 49 971

NCU02904 109 40 115 65 54 147

NCU04870 2 6 7059 218 261 3933

NCU05159 13 91 27383 1840 1333 7683

NCU09518 8 32 107 52 54 17

NCU09664 0.01 0.01 1039 4 4 130

NCU09924 2 19 112 43 45 2511

NCU03158 122 211 86 108 120 64

NCU07067 5 1242 225 426 368 121

NCU01353 1106 104 483 281 328 628

NCU07269 1 179 57 43 45 8

NCU06023 17 54 21 28 27 19

NCU06025 32 68 26 27 27 24

NCU00761 0.01 3 25 3 4 0.01

NCU06650 90 902 473 238 128 1654

NCU09416 0.01 0.01 1614 4 4 106

NCU00292 7 64 2892 2788 3721 4938

NCU03903 32 97 392 236 241 203

NCU04475 22 231 4395 2771 1833 2387

NCU06364 202 108 812 370 586 1635

NCU09575 20 632 228 245 222 95

NCU04230 250 1891 185 1237 538 80

NCU02366 3209 11379 4010 5034 5173 3702

NCU04280 1323 271 70 99 107 394

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU01378 88 574 235 436 374 186

NCU01861 0.01 394 138 717 593 497

NCU04583 237 103 47 381 409 42

NCU06616 19 636 149 853 329 45

NCU07325 3 1419 487 922 1047 177

NCU08771 28 3639 1648 5213 3274 485

NCU09553 201 574 217 918 509 74

NCU10055 3 812 330 851 856 321

NCU11289 2 26 5 14 17 0.01

NCU08750 33 213 1249 466 690 518

NCU08752 6 146 2469 450 1299 5930

NCU03049 5 106 43 58 55 35

NCU05653 49 62 21 39 27 16

NCU07133 12 1196 443 713 603 441

NCU08925 38 135 51 78 69 28

NCU09865 3 21 8 12 11 0.01

NCU11365 62 96 44 73 48 39

NCU07055 0.01 16 191 75 88 212

NCU07224 0.01 0.01 203 22 19 170

NCU01061 47 194 35 39 34 8

NCU03566 30 164 72 71 82 78

NCU04260 28 396 172 143 166 74

NCU05094 7 379 101 125 142 40

NCU05986 86 230 128 120 124 76

NCU06153 0.01 634 350 196 213 128

NCU09674 209 1068 454 452 424 601

NCU11241 265 492 158 225 194 128

NCU03013 452 1461 3723 3169 3589 4288

NCU05319 39 5 25 12 10 19

NCU04430 7 71 102 1738 599 45

NCU02059 520 83 1010 171 121 257

NCU00831 162 796 532 1450 897 559

NCU06055 161 151 347 273 246 233

NCU00263 55 118 158 670 324 191

NCU07200 215 477 667 4484 1939 312

NCU09992 25 177 420 3258 1302 143

NCU09265 1614 952 5131 1413 1691 2611

NCU00813 165 590 1841 555 639 598

NCU02455 1925 1985 5593 2466 2663 3092

NCU09223 1323 1972 8850 2804 2900 2955

NCU09485 444 248 940 299 324 489

NCU01648 838 418 1306 631 599 1003

NCU10497 738 602 1111 624 720 856

NCU00669 494 336 735 331 389 575

NCU021 18 66 74 45 62 59 28

NCU10762 149 47 123 80 67 161

NCU00244 126 117 59 56 60 58

NCU01068 135 371 1235 552 519 988 Table 1C: FPKMs identified from profiling data in different mutant strains and growth conditions

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU03319 940 1076 2513 1218 1267 1413

NCU08761 137 117 269 113 132 184

NCU01279 355 489 165 311 352 91

NCU03819 117 109 252 148 160 133

NCU08607 278 312 764 444 394 465

NCU09195 151 453 168 260 285 126

NCU07736 42 17 131 451 277 297

NCU01290 2430 43 74 20 29 681

NCU03396 2598 26 54 10 16 524

NCU09521 618 12 28 6 9 173

NCU03897 1005 305 611 239 291 569

NCU07746 448 249 969 303 300 424

NCU08897 1372 727 2550 971 1068 1948

NCU00169 486 315 1214 404 395 655

NCU02681 691 372 1150 435 391 519

NCU06333 488 307 1502 340 339 589

NCU01 146 1459 761 1819 1168 1149 1489

NCU00931 308 6 18 7 9 98

NCU07008 47 226 107 103 99 130

NCU03295 13 200 125 322 211 42

NCU07737 62 113 881 2349 1712 516

NCU08038 15 85 261 125 146 69

NCU02729 621 4 16 3 4 111

NCU03364 209 744 1586 596 647 654

NCU03817 66 135 46 62 62 58

NCU061 1 1 350 164 87 70 57 172

NCU081 15 47 48 396 97 185 82

NCU06931 9 48 33 75 53 14

NCU04077 1661 38 212 71 83 1471

NCU01862 10 369 103 229 239 31

NCU02795 61 24 53 47 52 64

NCU00812 459 103 208 88 131 217

NCU01856 850 504 2100 752 727 711

NCU03725 437 184 1312 322 317 701

NCU06971 10 336 1103 330 475 271

NCU07705 39 173 751 11 510 417

NCU08042 3 9 1262 15 0.01 16

NCU03643 21 198 57 246 163 24

NCU03043 147 1202 276 657 544 429

NCU05767 0.01 263 18 26 33 0.01

NCU00316 124 249 122 366 199 128

NCU00721 98 279 152 873 742 147

NCU07578 10 41 34 65 50 8

NCU04435 6 18 3 5 6 25

NCU05198 171 136 54 87 91 99

NCU10721 100 116 398 137 129 242

NCU11342 1 19 118 33 37 8

NCU00821 492 1869 1080 3808 2577 1577

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU09424 1 13 49 34 36 20

NCU09498 340 67 252 430 417 790

NCU09823 28 101 350 697 526 333

NCU09848 215 15 76 53 51 32

NCU10014 419 93 997 1874 1669 1536

NCU10039 8 56 888 376 441 167

NCU 10687 258 62 165 219 241 958

NCU00561 23 601 165 233 249 67

NCU00859 53 1441 184 112 162 50

NCU02042 70 164 98 40 51 106

NCU02164 75 909 287 346 343 105

NCU04482 7 70 9 19 10 42

NCU04486 18 449 157 152 193 58

NCU05236 7 386 120 147 141 77

NCU05761 0.01 16 0.01 0.01 0.01 0.01

NCU05763 0.01 18 0.01 0.01 0.01 0.01

NCU06328 63 517 97 45 48 124

NCU07948 11 206 28 33 41 28

NCU08140 0.01 5 0.01 0.01 0.01 0.01

NCU08447 7 144 39 44 40 16

NCU09734 13 653 25 43 53 24

NCU1201 1 0.01 30 2 3 4 0.01

NCU00408 245 33 79 19 26 73

NCU00633 464 87 244 67 116 141

NCU00870 58 103 7992 289 210 1281

NCU00871 0.01 3 19 3 3 0.01

NCU00965 410 321 1037 362 347 506

NCU01003 17 9 50 13 10 22

NCU01049 0.01 0.01 59 0.01 0.01 0.01

NCU01077 2 10 48 9 10 0.01

NCU01 148 28 27 106 37 36 36

NCU01944 58 20 441 46 47 43

NCU01970 96 1490 2320 719 761 1015

NCU01983 1113 36 184 35 43 171

NCU02008 90 108 235 61 65 100

NCU02061 24 17 66 0.01 2 39

NCU02600 3 8 26 8 9 0.01

NCU02625 21 45 150 44 45 21

NCU02720 419 27 79 21 28 106

NCU02915 34 77 818 153 142 74

NCU03152 317 89 325 103 95 180

NCU03329 91 170 1134 326 293 135

NCU03433 17 8 39 10 10 18

NCU04127 2828 968 3165 993 1099 1600

NCU04522 6 16 2283 22 102 45

NCU04830 532 66 381 109 80 292

NCU04905 347 354 1506 542 464 924

NCU05056 0.01 0.01 35 0.01 0.01 0.01

Gene Sucrose No Cellulose Aclr-l Aclr-2 Xylan

Carbon FPKM

NCU08383 0.01 1 15 16 16 8

NCU08491 0.01 1254 1223 1737 1330 63

NCU08634 4 28 58 91 78 18

NCU09075 10 0.01 6 23 10 0.01

NCU09415 84 10 213 86 96 248

NCU09856 6 26 102 103 74 93

NCU09874 306 303 696 4320 2036 525

NCU09906 47 38 151 256 206 363

NCU10284 488 46 318 259 154 462

NCU 10697 18 20 154 94 129 15

NCU1 1095 7 8 32 23 24 9

NCU1 1291 11 9 58 32 29 10

NCU1 1689 5 3 28 105 72 15

NCU11801 4 5 20 22 21 6

NCU1 1932 0.01 47 342 158 286 419

NCU00365 361 582 249 202 195 339

NCU00375 15 204 42 67 54 26

NCU00755 5 370 39 57 53 15

NCU01 109 0.01 122 14 23 25 0.01

NCU01292 16 26 14 13 12 19

NCU01551 78 423 168 176 193 57

NCU01649 395 416 153 217 170 293

NCU0301 1 1 98 4 7 4 0.01

NCU03417 67 297 88 109 105 41

NCU04285 1 19 5 6 4 0.01

NCU04843 546 2230 885 985 1378 371

NCU04851 44 91 42 51 50 54

NCU04861 3 61 16 21 22 8

NCU04862 49 668 245 247 294 91

NCU05006 99 1078 194 211 241 179

NCU05189 8 101 12 17 13 8

NCU05197 71 51 42 42 36 24

NCU05477 17 60 18 12 12 16

NCU05762 0.01 13 0.01 0.01 0.01 0.01

NCU05764 0.01 11 0.01 0.01 0.01 0.01

NCU05766 0.01 119 32 39 44 16

NCU05859 1 34 9 4 6 4

NCU05933 4 59 27 30 31 6

NCU06334 50 529 158 140 155 42

NCU07180 5 44 18 21 21 6

NCU07363 1208 5311 1592 2150 2283 1156

NCU08037 5 924 227 209 290 8

NCU08155 77 1590 216 354 221 192

NCU08156 13 275 43 45 40 23

NCU08170 7 33 16 15 14 21

NCU08455 20 1198 153 230 290 50

NCU08554 899 3675 1509 1940 1750 838

NCU08622 0.01 40 14 17 19 0.01

Table ID: Differential expression (DE) patterns between various mutant strains and growth conditions*

Gene Avicel v No Avicel v Avicel v Sucrose v Avicel v

Carbon DE Aclr-l DE Aclr-2 DE No Carbon Sucrose DE

DE

NCU00554 yes yes yes yes yes

NCU00944 cuffdiff yes cuffdiff cuffdiff

NCU01195 cuffdiff yes yes yes yes

NCU02785 yes cuffdiff cuffdiff yes yes

NCU02954 cuffdiff cuffdiff yes yes yes

NCU03131 yes cuffdiff cuffdiff cuffdiff cuffdiff

NCU04216 cuffdiff yes yes yes yes

NCU04298 yes cuffdiff cuffdiff yes yes

NCU04837 cuffdiff yes cuffdiff yes yes

NCU05548 yes yes yes yes cuffdiff

NCU07413 yes yes yes cuffdiff cuffdiff

NCU10283 yes yes yes yes cuffdiff

NCU00461 cuffdiff yes cuffdiff yes yes

NCU00591 yes yes yes yes yes

NCU00680 yes yes cuffdiff cuffdiff cuffdiff

NCU01402 cuffdiff yes yes yes yes

NCU02127 yes yes yes yes yes

NCU02704 cuffdiff yes yes yes yes

NCU02727 cuffdiff yes cuffdiff cuffdiff cuffdiff

NCU02936 yes yes yes yes cuffdiff

NCU03076 cuffdiff yes yes cuffdiff

NCU03415 yes yes cuffdiff yes yes

NCU12015 yes yes

NCU12015 yes

* Yes: Passed cuffdiff statistical test and was consistently different by a factor of 2 between all replicates of each condition. Cuffdiff: Passed cuffdiff statistical test but was not consistently different by a factor of 2 between all replicates tested.

Claims

CLAIMS What is claimed:

1. A method of degrading cellulose-containing material, the method comprising:

a) contacting cellulose-containing material with a fungal host cell comprising at least one recombinant nucleic acid encoding a transcription factor protein, wherein said transcription factor protein comprises a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187; and

b) incubating said fungal host cell and cellulose-containing material under conditions sufficient for the fungal host cell to degrade said cellulose-containing material.

2. The method of claim 1 , wherein said transcription factor protein comprises at least one additional polypeptide sequence selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

3. The method of claim 1 , wherein said transcription factor protein comprises at least two additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

4. The method of claim 1 , wherein said transcription factor protein comprises at least three additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

5. The method of claim 1 , wherein said transcription factor protein comprises SEQ ID NOs: 184, 185, 186, and 187.

6. The method of any one of claims 1 -5, wherein said fungal host cell is incubated under conditions sufficient for the fungal host cell to express said transcription factor protein.

7. The method of any one of claims 1 -6, wherein said fungal host cell produces a greater amount of one or more cellulases than a corresponding fungal host cell lacking said at least one recombinant nucleic acid.

8. The method of any one of claims 1 -7, wherein said cellulose-containing material comprises biomass.

9. The method of claim 8, wherein said biomass is subjected to pretreatment prior to being contacted with said fungal host cell.

10. The method of claim 9, wherein said pretreatment comprises one or more treatments selected from the group consisting of ammonia fiber expansion (AFEX), steam explosion, treatment with high temperature, treatment with high pressure, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (1L), treatment with electrolyzed water, and treatment with phosphoric acid.

1 1. The method of any one of claims 8-10, wherein said biomass comprises a plant material.

12. The method of claim 1 1 , wherein said plant material is selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, and energy cane.

13. The method of any one of claims 1 -12, wherein said fungal host cell further comprises one or more recombinant nucleic acids that encode a polypeptide involved in a biochemical pathway for the production of at least one biofuel.

14. The method of claim 13, further comprising incubating said fungal host cell with said degraded cellulose-containing material under conditions sufficient for the fungal host cell to convert the cellulose-containing material to at least one biofuel.

15. The method of claim 13 or claim 14, wherein said biofuel is selected from the group consisting of ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-l -butanol, 2-methyl- l - butanol, 3-methyl-l -pentanol, and octanol.

16. The method of any one of claims 1 -12, wherein said degraded cellulose-containing material is cultured with a fermentative microorganism under conditions sufficient to produce at least one fermentation product from the degraded cellulose-containing material.

17. A method of increasing the production of one or more cellulases from a fungal cell, the method comprising:

(a) providing a fungal host cell comprising at least one recombinant nucleic acid encoding a transcription factor protein, wherein said transcription factor protein comprises a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from the group consisting of SEQ ID NOs: 184, 1 85, 186, and 187; and

(b) culturing said host cell under conditions sufficient to support the expression of said at least one recombinant nucleic acid, wherein said fungal host cell produces a greater amount of said one or more cellulases than a corresponding host cell lacking said at least one recombinant nucleic acid.

18. The method of claim 17, wherein said fungal host cell is cultured in the absence of cellulose.

19. The method of any one of claims 1 -18, wherein said at least one recombinant nucleic acid is SEQ ID NO: 5.

20. The method of any one of claims 1 -19, wherein said at least one recombinant nucleic acid is operatively linked to a promoter selected from group consisting of ccg-1 and gpdA.

21. The method of any one of claims 1 -20, wherein said fungal host cell further comprises at least one additional recombinant nucleic acid encoding an additional transcription factor protein, wherein said additional transcription factor protein comprises a zinc(2)-cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191 , and 192.

22. The method of claim 21 , wherein said additional transcription factor protein comprises at least one additional polypeptide sequence selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, and 192.

23. The method of claim 21 , wherein said additional transcription factor protein comprises at least two additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191 , and 192.

24. The method of claim 21, wherein said additional transcription factor protein comprises at least three additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191 , and 192.

25. The method of claim 21, wherein said additional transcription factor protein comprises at least four additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 188, 189, 190, 191, and 192.

26. The method of claim 21 , wherein said additional transcription factor protein comprises SEQ ID NOs: 188, 189, 190, 191 , and 192.

27. The method of any one of claims 21 -26, wherein the at least one additional recombinant nucleic acid encoding said additional transcription factor protein is SEQ ID NO: 2.

28. The method of any one of claims 21 -27, wherein said at least one additional recombinant nucleic acid is operatively linked to a promoter selected from group consisting of ccg-1 and gpdA.

29. The method of any one of claims 1 -28, wherein said fungal host cell further comprises at least one recombinant nucleic acid encoding a hemicellulase.

30. The method of any one of claims 1 -29, wherein said fungal host cell is selected from the group consisting of Neurospora crassa, Metarhizium anisopliae, Gibberella zeae, Nectria haematococca, Magnaporthe oryzae, Neurospora tetrasperma, Sordaria macrospora,

Chaetomium globosum, Podospora anserina, Verticillium albo-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Penicillium chrysogenum, Leptosphaeria maculans, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Pyrenophora teres, Penicillium marneffei, Talaromyces stipitatus, Trichoderma reesei, Uncinocarpus reesii, Coccidioides immitus, Coccidioides posadasii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Sporotrichum ihermophile (Myceliophthora thermophila) , Thielavia terrestris- thermophilic, Acremonium cellulolyticus, Yarrowia lipolylica, Hansenula polymorpha,

Kluyveromyces lactis, Pichia pastoris, Mycosphaerella graminicola, Neosartorya fischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), and Chrysosporium lucknowense.

31. A method of reducing the viscosity of a pretreated biomass material, the method comprising contacting pretreated biomass material with a fungal host cell comprising at least one recombinant nucleic acid encoding a transcription factor protein, to yield a pretreated biomass material having reduced viscosity, wherein said transcription factor protein comprises a zinc(2)- cysteine(6) binuclear cluster domain, a PFAM04082 transcription factor domain, and at least one polypeptide sequence selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

32. The method of claim 31, wherein said transcription factor protein comprises at least one additional polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 84, 185, 186, and 187.

33. The method of claim 31 , wherein said transcription factor protein comprises at least two additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

34. The method of claim 31 , wherein said transcription factor protein comprises at least three additional polypeptide sequences selected from the group consisting of SEQ ID NOs: 184, 185, 186, and 187.

35. The method of claim 31 , wherein said transcription factor protein comprises SEQ ID NOs: 184, 185, 186, and 187.

The method of any one of claims 31 -35, wherein said at least one recombinant nucleic SEQ ID NO: 5.