# Patent

## Title
Transcription factors for cellulosic enzyme production

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase patent application of PCT/US2012/047898, filed Jul. 23, 2012, which claims the benefit of U.S. Provisional Application No. 61/510,466, filed Jul. 21, 2011, which are hereby incorporated by reference, in their entireties.


SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE
The content 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: 416272011100SubSeqList.txt, date recorded: Mar. 29, 2016, size: 995 KB).
FIELD
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
Liquid fuels derived from biomass 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.
The greatest advances in cellulase 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 cellulase production. Without a fundamental understanding of how particular mutations improve cellulase 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
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.
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, 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. 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.
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: 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.
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: 186 and 187.
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.
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.
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-butanol, 2-methyl-1-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.
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: 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 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.
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.
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.
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.
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.
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.
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: 188, 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: 119, 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.
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 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), and Chrysosporium lucknowense. 
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: 119, or SEQ ID NO: 183.
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: 119, or SEQ ID NO: 183.
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, 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.
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.
In some embodiments, provided herein is a fungal host cell containing at least one recombinant nucleic acid encoding a clr-2 transcription factor protein.
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.
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.
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: 119, or SEQ ID NO: 183.
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: 119, or SEQ ID NO: 183.
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.
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.
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 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, Neosartoryafischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense. 
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, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillusfumigatus, 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. 
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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-atrum, Glomerella graminicola, Grosmannia clavigera, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillusfumigatus, 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. 
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. 
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.
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.
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.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
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.
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.
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
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FIG. 1 depicts expression patterns for secreted enzymes after media shift. FIG. 1A depicts typical expression patterns for secreted enzymes after shift to no carbon or a new carbon source. FIG. 1B depicts message abundance for 16 predicted Neurospora crassa cellulases after cultures are shifted to no carbon or a new carbon source. FIG. 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.
FIG. 2A depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and sucrose cultures at 1 hour after transfer. FIG. 2B depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between sucrose and no-carbon at 1 hour. FIG. 2C depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and no-carbon at 1 hour. FIG. 2D depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between cellulose and sucrose at 4 hours. FIG. 2E depicts transcript abundance for the full Neurospora crassa (N. crassa) genome as compared between sucrose and no-carbon at 4 hours. FIG. 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.
FIG. 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. FIG. 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 16 hrs, 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. FIG. 3B depicts a comparison of the RNAseq derived differentially expressed gene lists from FIG. 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.
FIG. 4 depicts growth and enzyme secretion of deletion strains for cdr-1 (clr-1) and cdr-2 (clr-2). FIG. 4A depicts growth of wild type and deletion strains in 5 ml tubes with SMM, XMM or CMM. FIG. 4B depicts growth on a layer of saturated cellulose minimal medium. FIG. 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 Δclr-1 deletion strain grown on sucrose; lane 4 shows the results of the Δclr-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 Δclr-1 deletion strain grown on xylan; lane 7 shows the results of the Δclr-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 Δclr-1 deletion strain grown on Avicel®; and lane 10 shows the results of the Δclr-2 deletion strain grown on Avicel®. FIG. 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. FIG. 4E depicts total xylanase activity as measured by reducing sugars released from xylan from CMM or XMM cultures from FIG. 4D. FIG. 4F depicts total protein as measured by the Bradford assay in CMM or XMM cultures from FIG. 4D.
FIG. 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. FIG. 5B depicts the construct design for natively tagged cdr-1-GFP tagging and a mis-expression cdr-1 construct (under regulation of the ccg-1 promoter). FIG. 5C depicts expression profiles of cdr-1 and cdr-2 following shift of a SMM-grown culture to CMM. FIG. 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. FIG. 5E depicts the nuclear localization of natively GFP tagged CDR-1 (CLR-1). FIG. 5F depicts the relative expression of cbh-1 (NCU07340) and cdr-2 in the ccg1::cdr-1 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.
FIG. 6 depicts maximum likelihood phylogenetic trees of cdr-1 (clr-1) and cdr-2 (clr-2). FIG. 6A depicts cdr-1. FIG. 6B depicts cdr-2.
FIG. 7 depicts altered expression profiles in cdr (clr) deletion mutants. FIG. 7A depicts transcript abundance of predicted cellulase genes in wild type and cdr mutant strains at 4 hrs after transfer to CMM. FIG. 7B depicts expression profiles of predicted hemicellulase genes in wild type and cdr mutant strains at 4 hrs after transfer to CMM. FIG. 7C depicts global expression in Δcdr-1 (Δclr-1) as compared to wild type after transfer to CMM for 4 hrs. FIG. 7D depicts global expression in Δcdr-2 (Δclr-2) as compared to wild type after transfer to CMM for 4 hrs. FIG. 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. FIG. 7F depicts major classes of genes in the clusters from FIG. 7E. FIG. 7G depicts FPKM of selected cellulases and hemicellulases. Predicted hemicellulases exhibiting cellulase-like expression patterns are regulated by cdr-1 and cdr-2.
FIG. 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.
FIG. 9 depicts phylogenetic trees based on Bayesian inference. FIG. 9A depicts a cdr-1 (clr-1) tree. FIG. 9B depicts a cdr-2 (clr-2) tree.
FIG. 10 depicts transcript abundance of cbh-1 and cdt-2 in triple beta-glucosidase deletion mutants (ΔBG) with or without deletion of clr-1 or clr-2 four hours after shift to 0.2% cellobiose.
FIG. 11A 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. FIG. 11B depicts transcription of cbh-1 as a function of clr-1 abundance. All measurements are by RT-PCR 4 hours after media shift from sucrose cultures.
FIG. 12A depicts transcript abundance of cbh-1 relative to clr-2 in N. crassa strains 4 hours after shift from sucrose media. FIG. 12B depicts CMCase activity in WT and clr-2 mis-expression strain supernatants after growth in sucrose or Avicel®. FIG. 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®. FIG. 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®.
FIG. 13A depicts an SDS-PAGE gel of culture supernatants from WT and clr-2 mis-expression strains. FIG. 13B depicts transcript abundance (RNAseq) of selected cellulase genes in WT N. crassa, deletion strains for clr-1 and clr-2 after transfer to Avicel®; and WT and clr-2 mis-expression strains after transfer to no carbon.
FIG. 14 depicts hierarchical clustering of the N. crassa Avicel® regulon by FPKM in alternative inducing conditions and clr mutants.
FIG. 15 depicts a Western blot (anti-V5 antibody) of tagged and untagged clr-1 (NCU07705) in N. crassa lysates 4 hours after media shift to various carbon sources. Suc 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.
FIG. 16A depicts a Venn Diagram comparing the clr-1/2 ChiPseq regulons to the cellulose response RNAseq regulon. FIG. 16B depicts a graphical representation of the CLR-1 ChIP-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). FIG. 16C depicts CLR-1 and CLR-2 ChIP-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 of xlr-1 in nearly identical places.
FIG. 17 depicts the phenotype of A. nidulans clr deletion strains ΔclrA and ΔclrB. FIG. 17A depicts the enzyme activity of culture supernatants from ΔclrA and ΔclrB mutants grown on glucose and then shifted to Avicel® media. FIG. 17B depicts the total protein in supernatants of cultures grown on Avicel®. FIG. 17C depicts mycelial dry weights from WT and clr mutants from cultures on glucose and cellobiose (0.5% wt/vol). FIG. 17D depicts induction of selected cellulase genes in WT and the cdr 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. FIG. 17E depicts the expression of clrA and clrB in Aspergillus nidulans ΔclrA and ΔclrB 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.
FIG. 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. FIG. 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. FIG. 18C depicts the growth and CMCase activity of clrB mis-expression strain grown on cellobiose for 48 hrs.
FIG. 19 depicts growth of an N. crassa clrA mis-expression strain and an N. crassa clrB mis-expression strain on cellobiose and Avicel®. FIG. 19A depicts biomass accumulation of the clrA mis-expression strain on cellobiose. FIG. 19B depicts growth of the clrA mis-expression strain on Avicel®. FIG. 19C depicts biomass accumulation of the clrB mis-expression strain on cellobiose. FIG. 19D depicts growth of the clrB mis-expression strain on Avicel®.
FIG. 20A depicts the Clr-1 DNA-binding motif (SEQ ID NO: 239) as predicted from chromatin immunoprecipitation (ChIP) peaks. FIG. 20B depicts the Clr-2 DNA-binding motif (SEQ ID NO: 240) as predicted from chromatin immunoprecipitation (ChIP) peaks.
FIG. 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 depicted at amino acid residues 435-760 of the consensus sequence shown at the bottom of FIG. 21. The sequence alignment included the following sequences: Gibberella_zeae_PH-1 (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_CBS_148.51 (SEQ ID NO: 197), Podospora_anserina_S_mat+(SEQ ID NO: 198), Verticillium_albo-atrum_VaMs. 102 (SEQ ID NO: 199), Glomerella_graminicola_M1.001 (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_kw1407 (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._teres_0-1 (SEQ ID NO: 210), Leptosphaeria_maculans_JN3 (SEQ ID NO: 211), Talaromyces_stipitatus_ATCC_10500 (SEQ ID NO: 212), NCU00808 (SEQ ID NO: 213), and Trichoderma_reesei_clr-1 protein (SEQ ID NO: 182). A consensus sequence (SEQ ID NO: 241) is shown at the bottom of FIG. 21.
FIG. 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 depicted at amino acid residues 368-555 of the consensus sequence shown at the bottom of FIG. 22. The sequence alignment included the following sequences: AN6832 (SEQ ID NO: 214), Penicillium_marneffei_ATCC_18224 (SEQ ID NO: 215), Talaromyces_stipitatus_ATCC_10500 (SEQ ID NO: 216), AN3369 (SEQ ID NO: 217), Aspergillus_niger_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_M10.001 (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.102 (SEQ ID NO: 230), Pyrenophora_tritici-repentis_Pt-1C-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). A consensus sequence (SEQ ID NO: 242) is shown at the bottom of FIG. 22.



DETAILED DESCRIPTION
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.
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.
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.
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.
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.
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.
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.
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
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.
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
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.
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) (Stricker et al., App. Micro. Biotech., 78: 211-220 (2008)). Clr-2 is also a member of this superfamily.
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 R N, 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.
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-K-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).
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-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-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-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 (FIG. 22).
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-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).
Clr-1 polypeptides of the present disclosure include, without limitation, the polypeptide sequences 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), 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, 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).
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.
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.
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 of N. 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
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.
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
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.
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) (Stricker A R, et al., App. Micro. Biotech., 78: 211-220 (2008)). Clr-1 is also a member of this superfamily.
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 R N, 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.
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: 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-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).
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), 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), 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), EGO59545.1 (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_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), and EFQ30604.1 (SEQ ID NO: 68).
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 of N. 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.
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.
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
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, Ile, or Leu]-Thr-Phe-[Gln or Glu]. These conserved motifs can be used to identify further clr-2 transcription factors.
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
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-2 are 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.
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.
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
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.
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
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), 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, 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).
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: 119. 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.
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: 2, SEQ ID NO: 119, 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: 119, or SEQ ID NO: 183.
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.
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 18, or at least 20 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 24, or SEQ ID NO: 182.
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
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), XP_001910304.1 (SEQ ID NO: 71), XP_001223809.1 (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), 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), EGO59545.1 (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_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), and EFQ30604.1 (SEQ ID NO: 68).
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.
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.
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.
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: 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.
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
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.
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.
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
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
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)).
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.
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)).
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
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.
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.
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.
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 4:11-17 (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).
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, version10.3.0 (Invitrogen, Carlsbad, Calif.) 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 8:155-65 (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
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, 73-111 (1985)).
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: 507-511, (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.
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).
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 dodecylsulfate (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.
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 Tm−5° C. to Tm−20° C., moderate stringency at Tm−20° C. to Tm−35° C. and low stringency at Tm−35° C. to Tm−50° C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C. below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm−25° C. for DNA-DNA duplex and Tm−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.
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 (Tm) for the specific sequence at a defined ionic strength and pH.
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: 6×SSC and 1% SDS at 65° C.; 50% formamide, 4×SSC at 42° C.; 0.5×SSC to 2.0×SSC, 0.1% SDS at 50° C. to 65° C.; or 0.1×SSC to 2×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×SSC, and with, for example, a subsequent wash step with 0.2×SSC and 0.1% SDS at 65° C. for 10, 20 or 30 minutes.
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).
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 0.2×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×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.
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
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.
“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.
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.
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.
In some aspects, the host cell is fungus of the Ascomycota phylum. In some aspects, the host cell 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. 
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 fuckeliana, Aspergillus clavatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, 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, Neosartoryafischeri, Thermomyces lanuginosus (Humicola brevis, Humicola brevispora, Humicola grisea, Humicola lanuginosa, Monotospora lanuginosa, Sepedonium lanuginosum), Talaromyces thermophilus (Talaromyces dupontii, Penicillium dupontii), or Chrysosporium lucknowense. 
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.
“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.
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
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 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.
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.
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 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 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), 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, 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), or Trichoderma reesei clr-1 (SEQ ID NO: 182).
In some aspects, host cells contain a recombinant nucleic acid having the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 119, SEQ ID NO: 183, or any of SEQ ID NOs: 98-132.
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), 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), 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), EGO59545.1 (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_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), or EFQ30604.1 (SEQ ID NO: 68).
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.
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: 119, or SEQ ID NO: 183, and SEQ ID NO: 5 or SEQ ID NO: 165.
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.
Host cells of the disclosure may further contain one or more recombinant nucleic acid sequences encoding a hemicellulase. Hemicellulases include, without limitation, exoxylanases, endoxylanases, □-arabinofuranosidases, □-glucuronidases, □-xylosidases, and acetyl xylan esterases.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and/or octanol.
Methods of Producing and Culturing Host Cells of the Disclosure
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.
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.
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.
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.
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.
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.
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.
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.
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-I (glyceraldehyde 3-phosphate dehydrogenase-1) 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-I (transcription elongation factor) highly constitutive promoter; and the N. crassa xlr-1 (XlnR homolog) promoter, which is used frequently in Aspergillus species.
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.
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).
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.
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.
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.
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
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.
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
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.
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).
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.
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)
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).
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
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.
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.
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. 2003/0221211.
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.
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: 110-119, (2001); Fire et al., Nature 391: 806-811, (1998); and Timmons and Fire, Nature 395: 854, (1998).
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; 6,511,824; and 6,489,127).
In some aspects, RNAi sequences used herein correspond to a portion of SEQ ID NO: 2, SEQ ID NO: 119, 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 ID NO: 119, 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 ID NO: 119, SEQ ID NO: 183, SEQ ID NO: 5, or SEQ ID NO: 165.
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.
The term “target gene” or “target sequences”, refers to a gene targeted for reduced expression.
Antisense and Ribozyme Suppression
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
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.
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-I 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
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.
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. 
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
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.
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
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.
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 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.
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.
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.
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.
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.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
Methods for Reduction of the Viscosity of Pretreated Biomass Mixtures
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.
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.
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.
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.
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 (KCl), 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 KCl and/or ionic liquids, such as 1,3-dimethylimidazolium dimethyl phosphate ([DMIM]DMP) 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 110° 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
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 cdr-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.
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.
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.
Sugars that may be obtained from the degradation of cellulose-containing materials include, without limitation, glucose, cellobiose, xylose, arabinose, galactose, glucuronic acid, and mannose.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
Fermentative organisms include, without limitation, Saccharomyces spp.
Methods of Consolidated Bioprocessing
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.
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.
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.
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-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
Methods of Increasing the Production of Cellulases
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.
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.
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.
In other embodiments, the fungal host cell is cultured in the absence of cellulose.
Methods for increasing the growth rate of a cell disclosed herein apply to all host cells disclosed herein.
Methods of Producing Cellulases
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.
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.
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.
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.
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
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.
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.
Hemicellulases that may be produced by the methods provided herein include any enzyme having hemicellulose-degrading activity, including, without limitation, exoxylanases, endoxylanases, □-arabinofuranosidases, □-glucuronidases, □-xylosidases, and acetyl xylan esterases.
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 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.
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
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.
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
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 
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 FIG. 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.
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 (2011). 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 FIGS. 1B and 1C, 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.
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.
FIG. 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 FIG. 2D (4 hr) versus FIG. 2A (1 hr) (CMM versus SMM) and especially in the upper portion of FIG. 2F (4 hr) versus FIG. 2C (1 hr) (CMM versus NC). These 552 differentially expressed genes depicted in FIG. 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 1A-1E; 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
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.
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 dr 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.
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-I, and xlnR (xyr-1) (Stricker et al., App. Micro Biotech., 78: 211-220 (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 FIG. 5A, clr-1 and clr-2 also contain the conserved zinc (2) cysteine (6) binuclear domain, as well as a conserved central PFAM04082 domain.
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.
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.
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-1-gfp strain (FIG. 5F). However, when the ccg-1 clr-1-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
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
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 Δclr-1 or Δclr-2 CMM cultures (FIG. 7A). Thus wild type NC cultures and the Δclr-1 or Δclr-2 CMM culture appear to undergo an identical starvation response. When compared to wild type on CMM, it is clear that both Δclr-1 and Δclr-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 Δclr-1 or Δclr-2 mutants, with transcripts from some predicted hemicellulase genes showing wild type abundance in the Δclr-1 or Δclr-2 mutants, while others were dependent upon functional clr-1 and clr-2 for induction (FIG. 7B).
Global expression analyses in the Δclr-1 or Δclr-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
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 USA. 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).
We generated N. crassa mutant strains carrying deletions for the beta-glucosidase genes and for clr-1 or clr-2. When these ΔBG+Δclr mutants were switched from sucrose to cellobiose, the major cellulase cbh-1 was not induced (FIG. 10). These results strongly suggest that cellobiose, or a product derived therefrom, is the signal molecule that activates the clr-1/clr-2 pathway.
In contrast to cbh-1, the cellodextrin transporter cdt-2 is still strongly induced in the ΔBG Δclr-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
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.
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.
Our clr-1 mis-expression strain produces no detectable cellulase activity in sucrose culture. Results from a CMCase enzyme activity experiment are show in FIG. 11A. The results show that the enzyme activity was lower in the mis-expression mutant than in the wild-type strain (FIG. 11A). 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®.
Further, transcription of the major cellulase cbh-1 showed very poor correlation to clr-1 transcription in both wild type and mutant strains (FIG. 11B). 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
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. 12A). 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.
Transcriptional induction of cellulase genes by clr-2 mis-expression resulted in secretion of active cellulases (FIG. 12B). FIG. 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).
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). FIGS. 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/Pccg1-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/Pccg1-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.
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. 13A). 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).
RNAseq results from the clr-2 mis-expression strain complimented results from a wild-type (WT) N. crassa strain in various media conditions. The dr deletion strains on Avicel® and the ΔBG mutants on cellobiose illustrate several modes of transcriptional induction of WT strains on Avicel®. FIG. 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 Δclr-1 and Δclr-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 Δclr-1 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
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 FIG. 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-I 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
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 ChIPseq library and 318 genes in the CLR-2 library (FIG. 16A).
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 ChIP-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.
Overall, the ChIP-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.
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).
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 NCU11779 (FIG. 16B). However, as NCU11779 is not expressed in the 200 plus RNA-Seq experiments under a wide variety of conditions, we do not believe that NCU11779 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-I.
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
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 Δclr-1 and Δclr-2 mutants, the A. nidulans ΔclrA and ΔclrB 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 ΔclrB mutant, but the ΔclrA mutant showed  ̃50% of wild-type (WT) activity (FIGS. 17A and 17B). Both deletion mutants were deficient for growth on cellobiose, although ΔclrB was more strongly affected (FIG. 17C). Consistent with enzyme data, the induction pattern of major cellulase genes in the ΔclrB mutant was several thousand-fold less than WT (FIG. 17D). However, in the ΔclrA 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 ΔclrA 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.
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 ΔclrB 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 
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 ΔclrB 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 ΔclrB strain, with the A. fumigatus pyroA gene as a selective marker. FIG. 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 FIGS. 18B and 18C, 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 
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 Δclr-1 strain expressing clrA under the ccg-1 promoter was generated. The Δclr-1 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. 19A and 19B). A N. crassa Δclr-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 Δclr-2 on either Avicel® or cellobiose (FIGS. 19C and 19D). 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
The top 50 CLR-1 chromatin-immunoprecipitation peaks, which were identified by sequence analysis (ChIP-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 FIG. 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
The top CLR-2 chromatin-immunoprecipitation peaks, which were identified by sequence analysis (ChIP-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 FIG. 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 11 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 ChIP regulon with the predicted DNA binding motif, this number was increased to 84 with the simplified version of CCG(N11)CGG.
Example 14
CLR Protein Sequence Analysis
Clr-1
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.
As shown in FIG. 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).
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-I-Y-W-N-A-L-L-F-E-S-D-L-L-A-E-L-D-L-P-H-S-G-1-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.
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 acids 166-180 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 104-111 of the consensus sequence and has the following sequence: R-[NH]-[LM]-[ST]-[QP]-[STP]-[SP]-[DE] (SEQ ID NO: 192).
Clr-2
The N. crassa clr-2 amino acid sequence was aligned with 21 other clr-2 homologs 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.
As shown in FIG. 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 65-110 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.
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-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), where X can be any amino acid residue.
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 acids 614-619 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
The wild-type reference strain and background for all N. crassa mutant strains was FGSC 2489 (Neurospora crassa 74-OR23-1V A). Deletion strains for clr-1 and clr-2 with their open reading frames replaced by a hygromycin resistance cassette (FGSC 11029 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 A1149 (pyrG89; pyroA4; nkuA::argB) with knockout cassettes obtained from the Fungal Genetics Stock Center at the University of Missouri, Kansas City, Mo.
Transformants were crossed to LO1496 (fwA1, pyrG89, nicA2, pabaA1, from Berl R. Oakley Department of Molecular Biosciences, University of Kansas, Lawrence, Kans.) to remove nkuA::argB and pyroA4.
Culture Conditions for Media Shift Assay
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.).
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 4×106 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×g twice to remove mycelia and stored at 4° C. for analysis.
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 WHATMANTM 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)).
For enzyme activity assays, culture supernatants were sampled at 24 hours, filtered with WHATMANTM paper and stored at 4° C. for analysis within 72 hrs.
RNA Sequencing
RNA samples were reverse transcribed and prepared for high throughput sequencing with protocols adapted from Illumina Inc. Briefly, mRNA was purified with DYNABEADSTM Oligo dT magnetic beads (Illumina). 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. Illumina sequencing adapters were then ligated to the cDNA, 200 bp 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 Calif. Approximately 60 million single end 50 base-pair reads were obtained per library.
Analysis of Differential Expression
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 RNAseq library was analyzed.
Sequenced libraries were mapped against predicted transcripts from the N. crassa OR74A genome (version 10) with Bowtie (Langmead et 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., (2011) Genome Biology. 12:R22). The genes identified by Cuffdiffwere 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.
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 Δclr-1 and Δclr-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
To assess total cellulase activity, 500 μL of culture supernatant was incubated with 2.5 mg cellulose in 500 μL of 100 mM sodium acetate, pH 5 for 5 hours at 37° C. 40 μL of incubated sample was then added to 160 μL 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.
To assess hemicellulase activity, 100 μL of culture supernatant was incubated with 9 mg xylan in 900 μL 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.
Total protein was determined with the Bradford assay (BioRad).
Phylogenetic Analysis
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)).
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









Primers:



(SEQ ID NO: 7)










clr-1-F
5′-ATGACGCCGAACCGAGTG-3′



 









(SEQ ID NO: 8)










clr-1-R
5′-CAACAACACCAGAATGCGG-3′



 









(SEQ ID NO: 9)










clr-2-F
5′-TCCCGGCCATCAGACAGA-3′



 









(SEQ ID NO: 10)










clr-2-R
5′-ATCGGCACGGAAGGTTGTT-3′



 









(SEQ ID NO: 11)










B-actin-F
5′-TGATCTTACCGACTACCT-3′



 









(SEQ ID NO: 12)










B-actin-R
5′-CAGAGCTTCTCCTTGATG-3′



 









(SEQ ID NO: 13)










cbh1-F
5′-ATCTGGGAAGCGAACAAAG-3′



 









(SEQ ID NO: 14)










cbh1-R
5′-TAGCGGTCGTCGGAATAG-3′






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-1-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 K J and Schmittgen T D, 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





NCU00554
aspartate-semialdehyde
Amino Acid

No



dehydrogenase
Metabolism

Induction


NCU00944
l-allo-threonine aldolase
Amino Acid

No




Metabolism

Induction


NCU01195
Glu/Leu/Phe/Val dehydrogenase
Amino Acid

No




Metabolism

Induction


NCU02785
phospho-2-dehydro-3-
Amino Acid

No



deoxyheptonate aldolase
Metabolism

Induction


NCU02954
homoisocitrate dehydrogenase
Amino Acid

No




Metabolism

Induction


NCU03131
FAD dependent oxidoreductase
Amino Acid

No



superfamily
Metabolism

Induction


NCU04216
amidophosphoribosyltransferase
Amino Acid

No




Metabolism

Induction


NCU04298
pentafunctional AROM polypeptide
Amino Acid

No




Metabolism

Induction


NCU04837
mitochondrial 2-oxodicarboxylate
Amino Acid

No



carrier 1
Metabolism

Induction


NCU05548
phospho-2-dehydro-3-
Amino Acid

No



deoxyheptonate aldolase
Metabolism

Induction


NCU07413
cytosine deaminase, variant
Amino Acid

No




Metabolism

Induction


NCU10283
tryptophan synthetase
Amino Acid

No




Metabolism

Induction


NCU00461
NAD-specific glutamate
Amino Acid

No



dehydrogenase
Metabolism

Repression


NCU00591
methylcrotonoyl-CoA carboxylase
Amino Acid

No



subunit alpha
Metabolism

Repression


NCU00680
2-methylcitrate dehydratase
Amino Acid

No




Metabolism

Repression


NCU01402
indoleamine 2,3-dioxygenase
Amino Acid

No




Metabolism

Repression


NCU02127
methylcrotonoyl-CoA carboxylase
Amino Acid

No



subunit beta
Metabolism

Repression


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
delta-1-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


NCU10110
3-hydroxyisobutyrate
Amino Acid

WT



dehydrogenase
Metabolism

Induction


NCU03861
glutaminase A
Amino Acid

WT




Metabolism

Repression


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
No





Synthesis
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
No



dehydratase

Anabolism
Induction


NCU06189
5-aminolevulinate synthase
Anabolism
Heme
No





Synthesis
Induction


NCU05165
pyridoxamine phosphate oxidase
Anabolism
Vitamin
No





Metabolism
Induction


NCU04865
polyketide synthase 3
Anabolism

Partial






Repression


NCU05011
polyketide synthase 2
Anabolism

WT






Induction


NCU00762
endoglucanase 3
Carbon
Cellulases
No




Metabolism

Induction


NCU00836
hypothetical protein
Carbon
Cellulases
No




Metabolism

Induction


NCU01050
endoglucanase II
Carbon
Cellulases
No




Metabolism

Induction


NCU02240
endoglucanase II
Carbon
Cellulases
No




Metabolism

Induction


NCU02344
fungal cellulose binding domain-
Carbon
Cellulases
No



containing
Metabolism

Induction


NCU02916
endoglucanase II
Carbon
Cellulases
No




Metabolism

Induction


NCU03328
endoglucanase II
Carbon
Cellulases
No




Metabolism

Induction


NCU04854
endoglucanase EG-1
Carbon
Cellulases
No




Metabolism

Induction


NCU05057
endoglucanase EG-1
Carbon
Cellulases
No




Metabolism

Induction


NCU05121
endoglucanase V
Carbon
Cellulases
No




Metabolism

Induction


NCU07190
exoglucanase 3
Carbon
Cellulases
No




Metabolism

Induction


NCU07340
exoglucanase 1
Carbon
Cellulases
No




Metabolism

Induction


NCU07760
endoglucanase IV
Carbon
Cellulases
No




Metabolism

Induction


NCU07898
endoglucanase IV
Carbon
Cellulases
No




Metabolism

Induction


NCU08760
endoglucanase II
Carbon
Cellulases
No




Metabolism

Induction


NCU09680
exoglucanase 2
Carbon
Cellulases
No




Metabolism

Induction


NCU03322
GDSL family lipase
Carbon
Fatty Acid/
WT




Metabolism
Isoprenoid
Induction





Metabolism


NCU07362
L-lactate ferricytochrome c
Carbon
Fermentation
Partial



oxidoreductase
Metabolism

Repression


NCU03813
formate dehydrogenase
Carbon
Fermentation
WT




Metabolism

Induction


NCU04539
L-lactate dehydrogenase
Carbon
Fermentation
WT




Metabolism

Repression


NCU08687
galactokinase
Carbon
Galactose
No




Metabolism
Utilization
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-1,6-bisphosphatase
Carbon
Glycolysis
No




Metabolism

Repression


NCU00575
glucokinase
Carbon
Glycolysis
Partial




Metabolism

Induction


NCU04401
fructose-bisphosphate aldolase
Carbon
Glycolysis
WT




Metabolism

Induction


NCU02855
endo-1,4-beta-xylanase A
Carbon
Hemicellulases
No




Metabolism

Induction


NCU05924
endo-1,4-beta-xylanase
Carbon
Hemicellulases
No




Metabolism

Induction


NCU05955
Cel74a
Carbon
Hemicellulases
No




Metabolism

Induction


NCU07326
hypothetical protein
Carbon
Hemicellulases
No




Metabolism

Induction


NCU09775
alpha-N-arabinofuranosidase
Carbon
Hemicellulases
No




Metabolism

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-1,4-beta-xylanase 2
Carbon
Hemicellulases
WT




Metabolism

Induction


NCU08087
hypothetical protein
Carbon
Hemicellulases
WT




Metabolism

Induction


NCU08189
endo-1,4-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


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
Acr1
Carbon
Mitochondrial
WT




Metabolism
Carrier
Repression


NCU08398
aldose 1-epimerase
Carbon
Monnosaccharide
No




Metabolism
Metabolism
Induction


NCU10683
NRS/ER
Carbon
Monnosaccharide
No




Metabolism
Metabolism
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
No




Metabolism
Degredation
Induction


NCU04623
beta-galactosidase
Carbon
Oligosaccharide
No




Metabolism
Degredation
Induction


NCU04952
beta-D-glucoside glucohydrolase
Carbon
Oligosaccharide
No




Metabolism
Degredation
Induction


NCU05956
beta-galactosidase
Carbon
Oligosaccharide
No




Metabolism
Degredation
Induction


NCU07487
periplasmic beta-glucosidase
Carbon
Oligosaccharide
No




Metabolism
Degredation
Induction


NCU08755
beta-glucosidase 1
Carbon
Oligosaccharide
No




Metabolism
Degredation
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


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
WT




Metabolism
Phosphate
Induction





Pathway


NCU00206
cellobiose dehydrogenase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU00710
acetyl xylan esterase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU01059
glycosyl hydrolase family 47 protein
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU03181
acetylxylan esterase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU04494
acetyl xylan esterase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU05598
rhamnogalacturonase B
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU05751
cellulose-binding protein
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU08176
pectate lyase A
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU08746
starch binding domain-containing
Carbon
Polysaccharide
No



protein
Metabolism
Degradation
Induction


NCU08785
fungal cellulose binding domain-
Carbon
Polysaccharide
No



containing
Metabolism
Degradation
Induction


NCU09445
Cip2
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU09491
feruloyl esterase B
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU09582
chitin deacetylase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU09764
hypothetical protein
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU09774
cellulase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU09976
rhamnogalacturonan acetylesterase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU10045
pectinesterase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU11068
endo-beta-1,4-mannanase
Carbon
Polysaccharide
No




Metabolism
Degradation
Induction


NCU11198
arabinogalactan endo-1,4-beta-
Carbon
Polysaccharide
No



galactosidase
Metabolism
Degradation
Induction


NCU02904
alpha/beta hydrolase fold protein
Carbon
Polysaccharide
Partial




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-1,2-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
No




Metabolism
Lipases/Esterases
Induction


NCU06650
secretory phospholipase A2
Carbon
Secreted
No




Metabolism
Lipases/Esterases
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
WT




Metabolism
Utilization
Induction


NCU08384
xylose reductase
Carbon
Xylose
WT




Metabolism
Utilization
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
No



glucosyltransferase
Components
Synthesis or
Induction





Modifcation


NCU09425
NdvB protein
Cellular
Cell Wall
Partial




Components
Synthesis or
Induction





Modifcation


NCU02478
alpha-1,3-glucan synthase Ags2
Cellular
Cell Wall
WT




Components
Synthesis or
Induction





Modifcation


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



endoglucanase
Components
Synthesis or
Induction





Modifcation


NCU01689
mitochondrial DNA replication
Cellular
Mitochondrial
No



protein YHM2
Components
Reproduction
Induction


NCU11721
mitochondrial inner membrane
Cellular
Mitochondrial
No



protease subunit 2
Components
Reproduction
Induction


NCU02396
mitochondrial FAD-linked
Cellular
Mitochondrial
No



sulfhydryl oxidase
Components
Reproduction
Repression


NCU07481
morphogenesis protein
Cellular
Morphology
WT




Components

Induction


NCU03137
nuclear elongation and deformation
Classification

No



protein 1
Unclear

Induction


NCU02500
clock-controlled pheromone CCG-4
Clock

No




Controlled

Induction


NCU00565
lipoic acid synthetase
Cofactors

No






Repression


NCU02705
F1F0 ATP synthase assembly
Electron

No



protein Atp10
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

No



Regucalcin
Binding

Induction


NCU08691
EF-hand calcium-binding domain-
Ion

WT



containing
Binding

Repression


NCU09043
caleosin domain-containing protein
Lipid

Partial




Associated

Repression


NCU07432
tetraspanin
Membrane

Partial




Associated

Induction


NCU05841
UMTA
Methyl

Partial




transferase

Induction


NCU02361
formamidase
Nitrogen

No




and Sulfur

Repression




Metabolism


NCU10051
flavohemoglobin
Nitrogen

Partial




Metabolism

Induction


NCU04720
nitrite reductase
Nitrogen

WT




Metabolism

Induction


NCU04698
spermine/spermidine synthase
Nucleotide

Partial




Binding

Induction


NCU00177
phosphoribosylformylglycinamidine
Nucleotide

No



cyclo-ligase
Metabolism

Induction


NCU01786
ribose-phosphate
Nucleotide

No



pyrophosphokinase II
Metabolism

Induction


NCU03117
inosine-5′-monophosphate
Nucleotide

No



dehydrogenase IMD2
Metabolism

Induction


NCU05254
ribose-phosphate
Nucleotide

No



pyrophosphokinase
Metabolism

Induction


NCU03963
5′-methylthioadenosine
Nucleotide

No



phosphorylase
Metabolism

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-adenosylmethionine-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


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






Induction


NCU09992
serine peptidase
Protease

WT






Induction


NCU09265
calreticulin
Protein Synthesis
Protein
No




and Secretion
Folding
Induction


NCU00813
disulfide isomerase
Protein Synthesis
Protein
No




and Secretion
Folding
Induction


NCU02455
FKBP-type peptidyl-prolyl cis-trans
Protein Synthesis
Protein
No



isomerase
and Secretion
Folding
Induction


NCU09223
protein disulfide-isomerase
Protein Synthesis
Protein
No




and Secretion
Folding
Induction


NCU09485
related to stress protein ORP150
Protein Synthesis
Protein
No




and Secretion
Folding
Induction


NCU01648
dolichyl-phosphate-mannose-
Protein Synthesis
Protein
No



protein
and Secretion
Modification
Induction


NCU10497
oligosaccharyl transferase STT3
Protein Synthesis
Protein
No



subunit
and Secretion
Modification
Induction


NCU00669
oligosaccharyl transferase subunit
Protein Synthesis
Protein
No




and Secretion
Modification
Induction


NCU02118
palmitoyltransferase PFA4
Protein Synthesis
Protein
No




and Secretion
Modification
Repression


NCU10762
UDP-N-acetyl-glucosamine-1-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
No




and Secretion
Trafficing
Induction


NCU03319
COPII-coated vesicle protein
Protein Synthesis
Protein
No



SurF4/Erv29
and Secretion
Trafficing
Induction


NCU08761
vacuolar sorting receptor
Protein Synthesis
Protein
No




and Secretion
Trafficing
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-binding
Protein Synthesis
rRNA
No



protein CBF5
and Secretion
Production
Induction


NCU03396
nucleolar protein nop-58
Protein Synthesis
rRNA
No




and Secretion
Production
Induction


NCU09521
ribosome biogenesis protein
Protein Synthesis
rRNA
No




and Secretion
Production
Induction


NCU03897
RNA binding effector protein
Protein Synthesis
Translation
No



Scp160
and Secretion

Induction


NCU07746
F-box domain-containing protein
Protein Synthesis
Translocation
No




and Secretion

Induction


NCU08897
protein transporter SEC61 subunit
Protein Synthesis
Translocation
No



alpha
and Secretion

Induction


NCU00169
translocation complex componenet
Protein Synthesis
Translocation
No




and Secretion

Induction


NCU02681
translocation protein
Protein Synthesis
Translocation
No




and Secretion

Induction


NCU06333
translocation protein SEC62
Protein Synthesis
Translocation
No




and Secretion

Induction


NCU01146
signal sequence receptor alpha
Protein Synthesis
Translocation
Partial



chain
and Secretion

Induction


NCU00931
lysyl-tRNA synthetase
Protein Synthesis
tRNA
No




and Secretion
Charging
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

No




Transduction

Induction


NCU03364
DENN domain-containing protein
Signal

No




Transduction

Induction


NCU03817
FMI1 protein
Signal

Partial




Transduction

Repression


NCU06111
GTPase Ras2p
Signal

WT




Transduction

Repression


NCU08115
DNA mismatch repair protein
Stress

Partial



Msh3
Response

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 phd1
Transcriptional
Chromatin
WT




Regulation
Remodeling
Induction


NCU00812
exosome complex exonuclease
Transcriptional
RNA
No



RRP41
Regulation
processing
Induction


NCU01856
transcriptional activator hac1
Transcriptional
Transcription
No




Regulation
Factors
Induction


NCU03725
VIB-1
Transcriptional
Transcription
No




Regulation
Factors
Induction


NCU06971
transcriptional activator xlnR
Transcriptional
Transcription
No




Regulation
Factors
Induction


NCU07705
C6 finger domain-containing
Transcriptional
Transcription
No



protein
Regulation
Factors
Induction


NCU08042
fungal specific transcription factor
Transcriptional
Transcription
No




Regulation
Factors
Induction


NCU03643
cutinase transcription factor 1 beta
Transcriptional
Transcription
No




Regulation
Factors
Repression


NCU03043
C2H2 finger domain-containing
Transcriptional
Transcription
Partial



protein FlbC
Regulation
Factors
Repression


NCU05767
PRO1A C6 Zink-finger protein
Transcriptional
Transcription
WT




Regulation
Factors
Repression


NCU00316
peroxisomal adenine nucleotide
Transporter
Amino Acid
No



transporter 1

Transporter
Repression


NCU00721
proline-specific permease
Transporter
Amino Acid
No





Transporter
Repression


NCU07578
peroxisomal adenine nucleotide
Transporter
Amino Acid
No



transporter 1

Transporter
Repression


NCU04435
general amino acid permease AGP3
Transporter
Amino Acid
Partial





Transporter
Repression


NCU05198
general amino acid permease
Transporter
Amino Acid
Partial





Transporter
Repression


NCU10721
solute carrier family 35 member B1
Transporter
Carbohydrate
No



protein

Transport
Induction


NCU11342
MFS hexose transporter
Transporter
Carbohydrate
No





Transport
Induction


NCU00821
sugar transporter
Transporter
Carbohydrate
No





Transport
Repression


NCU08561
succinate/fumarate mitochondrial
Transporter
Carbohydrate
No



transporter

Transport
Repression


NCU09287
sugar transporter
Transporter
Carbohydrate
No





Transport
Repression


NCU00801
MFS lactose permease
Transporter
Carbohydrate
Partial





Transport
Induction


NCU00809
MFS monosaccharide transporter
Transporter
Carbohydrate
Partial





Transport
Induction


NCU07668
MFS multidrug transporter
Transporter
Carbohydrate
Partial





Transport
Induction


NCU05089
MFS monocarboxylate transporter
Transporter
Carbohydrate
Partial





Transport
Repression


NCU08152
high affinity glucose transporter
Transporter
Carbohydrate
Partial





Transport
Repression


NCU01633
hexose transporter HXT13
Transporter
Carbohydrate
WT





Transport
Induction


NCU04537
monosaccharide transporter
Transporter
Carbohydrate
WT





Transport
Induction


NCU05853
MFS sugar transporter
Transporter
Carbohydrate
WT





Transport
Induction


NCU08114
hexose transporter
Transporter
Carbohydrate
WT





Transport
Induction


NCU00023
ferric reductase
Transporter
Ion
No





Transporter
Induction


NCU02009
FreB
Transporter
Ion
No





Transporter
Induction


NCU07068
K(+)/H(+) antiporter 1
Transporter
Ion
No





Transporter
Repression


NCU03305
calcium-transporting ATPase
Transporter
Ion
Partial





Transporter
Induction


NCU08225
high affinity nickel transporter nic1
Transporter
Ion
Partial





Transporter
Repression


NCU08147
Na or K P-type ATPase
Transporter
Ion
Partial





Transporter
Repression


NCU06366
Ca2+/H+ antiporter
Transporter
Ion
WT





Transporter
Repression


NCU05585
MFS quinate transporter
Transporter
Quinate
Partial






Repression


NCU06138
quinate permease
Transporter
Quinate
WT






Induction


NCU05591
ABC transporter CDR4
Transporter
Trehalose
WT





Export
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

Partial




Membrane

Repression




Proteins


NCU00848
integral membrane protein TmpA
Unknown

WT




Membrane

Repression




Proteins


NCU00449
hypothetical protein
Unknown

No




Secreted

Induction


NCU00849
hypothetical protein
Unknown

No




Secreted

Induction


NCU01058
hypothetical protein
Unknown

No




Secreted

Induction


NCU01076
hypothetical protein
Unknown

No




Secreted

Induction


NCU01196
hypothetical protein
Unknown

No




Secreted

Induction


NCU01978
hypothetical protein
Unknown

No




Secreted

Induction


NCU02138
hypothetical protein
Unknown

No




Secreted

Induction


NCU03083
hypothetical protein
Unknown

No




Secreted

Induction


NCU03982
glucose-regulated protein
Unknown

No




Secreted

Induction


NCU04948
hypothetical protein
Unknown

No




Secreted

Induction


NCU05230
hypothetical protein
Unknown

No




Secreted

Induction


NCU05863
hypothetical protein
Unknown

No




Secreted

Induction


NCU05864
hypothetical protein
Unknown

No




Secreted

Induction


NCU06152
hypothetical protein
Unknown

No




Secreted

Induction


NCU06607
hypothetical protein
Unknown

No




Secreted

Induction


NCU08756
hypothetical protein
Unknown

No




Secreted

Induction


NCU08790
hypothetical protein
Unknown

No




Secreted

Induction


NCU09295
hypothetical protein
Unknown

No




Secreted

Induction


NCU09524
hypothetical protein
Unknown

No




Secreted

Induction


NCU11268
hypothetical protein
Unknown

No




Secreted

Induction


NCU11542
hypothetical protein
Unknown

No




Secreted

Induction


NCU11753
hypothetical protein
Unknown

No




Secreted

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


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




Secreted

Induction


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


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


No



protein


Induction


NCU02625
hypothetical protein


No






Induction


NCU02720
hypothetical protein


No






Induction


NCU02915
hypothetical protein


No






Induction


NCU03152
DUF1348 domain-containing


No



protein


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


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






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


NCU08397
hypothetical protein


Partial






Induction


NCU08748
hypothetical protein


Partial






Induction


NCU08867
hypothetical protein


Partial






Induction


NCU09176
hypothetical protein


Partial






Induction


NCU11769



Partial






Induction


NCU11828



Partial






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






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 52A11


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


NCU04928
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






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






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






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





Annotation: Broad Institute Annotation


Group 1: Author's hand curated annotation category


Group 2: Author's hand curated annotation/function













TABLE 1B







Annotation information for genes in Table 1A

















Signal P
TM

Tian et
Tian et al.


Gene
CAZy
Signal P
Confidence
Domains
LCMS
al., MA
Annotation

















NCU00554

Mitochondrion
4






NCU00944

Mitochondrion
2


NCU01195

Other
1


NCU02785

Other
2


NCU02954

Other
2


NCU03131

Mitochondrion
1


NCU04216

Other
5


NCU04298

Other
2


NCU04837

Other
4


NCU05548

Other
2


NCU07413

Other
1


NCU10283

Other
5


NCU00461

Other
2


NCU00591

Mitochondrion
1


NCU00680

Mitochondrion
1


NCU01402

Other
1


NCU02127

Mitochondrion
2


NCU02704

Mitochondrion
1


NCU02727

Mitochondrion
2


NCU02936

Mitochondrion
2


NCU03076

Mitochondrion
1


NCU03415

Other
1


Avi/Mis


NCU03648

Secretory
2




Pathway


NCU03913

Mitochondrion
2


NCU05499

Other
2


NCU05537

Other
2


NCU05977

Other
2


NCU06448

Mitochondrion
1


NCU06543

Mitochondrion
1


NCU07153

Mitochondrion
2


NCU08216

Other
1


NCU09116

Other
2


NCU09266

Mitochondrion
1


NCU09864

Mitochondrion
1


NCU11195

Mitochondrion
3


NCU01830

Other
2


NCU02126

Mitochondrion
2


NCU01744

Other
1


NCU03748

Other
4


NCU06625

Other
3


NCU04130

Other
5


NCU10110

Secretory
5
1




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


NCU05011

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


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
Hemicellulase




Pathway


NCU05924
GH10
Secretory
2

Avi/Mis
Avi/Mis
Hemicellulase




Pathway


NCU05955
CBM1,
Secretory
2

Avi/Mis
Avi/Mis
Hemicellulase



GH74
Pathway


NCU07326
GH43
Secretory
1

Avi/Mis
Avi/Mis
Hemicellulase




Pathway


NCU09775
GH54
Secretory
1

Mis
Mis
Hemicellulase




Pathway


NCU04997
CBM1,
Secretory
2



Hemicellulase



GH10
Pathway


NCU01900
GH43
Other
2


Avi/Mis
Hemicellulase


NCU02343
GH51
Secretory
1

Mis
Avi/Mis
Hemicellulase




Pathway


NCU07225
CBM1,
Secretory
2

Avi/Mis
Avi/Mis
Hemicellulase



GH11
Pathway


NCU08087
GH26
Other
2



Hemicellulase


NCU08189
GH10
Secretory
1

Avi/Mis
Avi/Mis
Hemicellulase




Pathway


NCU09652
GH43
Other
2


Avi/Mis
Hemicellulase


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


NCU07719

Other
2


NCU12093

Other
2


NCU05818

Other
3


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
CBM1
Secretory
3

Avi/Mis
Avi/Mis




Pathway


NCU00710
CBM1,
Secretory
2


Mis



CE1
Pathway


NCU01059
GH47
Secretory
3
1




Pathway


NCU03181

Secretory
3


Avi/Mis




Pathway


NCU04494
CE1
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
CE1
Secretory
1

Mis
Avi/Mis




Pathway


NCU09445
CE15
Secretory
1




Pathway


NCU09491
CE1
Secretory
2

Avi/Mis
Mis




Pathway


NCU09582
CE4
Secretory
1


Avi/Mis




Pathway


NCU09764
CBM1
Secretory
1


Avi/Mis




Pathway


NCU09774
CE1
Secretory
1


Avi




Pathway


NCU09976
CE12
Secretory
4


Mis




Pathway


NCU10045
CE8
Secretory
1


Avi/Mis




Pathway


NCU11068

Other
3


NCU11198
GH53
Secretory
1




Pathway


NCU02904

Secretory
1




Pathway


NCU04870
CE1
Secretory
1

Mis
Mis




Pathway


NCU05159
CBM1,
Secretory
1

Mis
Avi/Mis



CE5
Pathway


NCU09518

Secretory
1




Pathway


NCU09664
CE5
Secretory
1


Avi/Mis




Pathway


NCU09924
GH93
Secretory
1


Mis




Pathway


NCU03158

Secretory
4
1




Pathway


NCU07067
GH47
Secretory
1
3




Pathway


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


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


NCU11721

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


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


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


NCU10497

Other
3
13


NCU00669

Secretory
1
1




Pathway


NCU02118

Secretory
3
4




Pathway


NCU10762

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




Pathway


NCU00169

Other
3
3


NCU02681

Secretory
1
1




Pathway


NCU06333

Other
2
2

Mis


NCU01146

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


NCU06111

Mitochondrion
5


NCU08115

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


NCU10721

Other
4
8


NCU11342

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


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


NCU10009

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




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


NCU11268

Secretory
4




Pathway


NCU11542

Secretory
1




Pathway


NCU11753

Secretory
2




Pathway


NCU00175

Secretory
3




Pathway


NCU00250

Secretory
1
2




Pathway


NCU00322

Secretory
2




Pathway


NCU00695

Secretory
3


Avi




Pathway


NCU07311

Secretory
1
4




Pathway


NCU08171

Secretory
2




Pathway


NCU08521

Secretory
1
2




Pathway


NCU10507

Secretory
1




Pathway


NCU07143

Secretory
1

Avi/Mis
Avi/Mis




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


NCU11957

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




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


NCU10687

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


NCU12011

Secretory
1




Pathway


NCU00408

Other
1


NCU00633

Other
2


NCU00870

Other
4


Avi/Mis


NCU00871

Other
2


NCU00965

Other
2
2


NCU01003

Other
3


NCU01049

Other
3


NCU01077

Other
1


NCU01148

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


NCU08113

Other
2


NCU08117

Other
4


NCU08379

Mitochondrion
4
1


NCU08624

Other
3
6


NCU08784

Other
3


NCU09003

Other
5


NCU09426

Other
4


NCU09479

Other
3
1


NCU09522

Other
5


NCU09523

Other
4


NCU09689

Other
1


Avi/Mis


NCU10521

Other
5


NCU11118

Mitochondrion
3


NCU11278

Other
3


NCU11327


NCU11397


NCU11690

Other
3


NCU11722


NCU11862

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


NCU10276

Other
2
11


NCU11697


NCU11944


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


NCU04904

Other
3


NCU05351

Mitochondrion
4


NCU05501

Other
2


Mis


NCU05906

Other
2


NCU06373

Mitochondrion
5


NCU07270

Other
2


NCU08116

Other
5


NCU08397

Other
1
11

Avi/Mis


NCU08748

Other
1
11


NCU08867

Other
5
1


NCU09176

Other
3


NCU11769


NCU11828


NCU11905


NCU00011

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


NCU09115

Other
3
1


NCU09883

Mitochondrion
5
7

Avi


NCU10658

Other
2


NCU10770

Other
2


NCU11294


NCU00304

Mitochondrion
3


Mis


NCU00798

Mitochondrion
5

Avi
Avi


NCU01136

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


NCU10697

Mitochondrion
2


NCU11095

Other
2


NCU11291

Other
4


NCU11689

Other
4


NCU11801


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


NCU03011

Mitochondrion
3


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


NCU11774


NCU11881

Other
5


NCU11974

Other
2


NCU11989

Other
2


NCU12012


NCU12014

Other
4


NCU12015

Other
4





CAZy: Predicted Domains from the Carbohydrate Active Enzymes database (Cantarelet al., (2009) Nucleic Acids Res 37: D233-238 [PMID: 18838391]).


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


TM Domains: Predicted Transmembrane Domains LCMS: Condition under which a gene product was detected in the culture supernatant by Tian et al., Proc Natl Acad Sci 2009.


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


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



















Xylan


Gene
Sucrose
No Carbon
Cellulose
Δclr-1
Δclr-2
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


NCU10283
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


NCU05537
14
465
224
1257
813
32


NCU05977
11
762
100
486
310
761


NCU06448
944
1508
978
2366
1474
722


NCU06543
151
784
414
1264
762
166


NCU07153
738
2886
1877
6968
4284
718


NCU08216
340
187
80
421
251
67


NCU09116
376
206
82
417
229
62


NCU09266
101
1296
603
2306
1479
379


NCU09864
40
1090
416
2430
1372
52


NCU11195
166
677
307
598
450
261


NCU01830
9
645
446
1362
1859
11


NCU02126
7
383
117
687
407
40


NCU01744
1187
197
500
461
264
1148


NCU03748
5279
95
245
130
178
1873


NCU06625
41
400
61
112
113
70


NCU04130
33
155
375
612
304
350


NCU10110
8
56
1068
615
786
2096


NCU03861
3
31
5
5
8
0.01


NCU07623
1
69
29
30
28
9


NCU01427
170
1652
490
537
591
196


NCU03651
256
51
979
243
54
809


NCU02579
27
1820
878
1457
1443
181


NCU07307
1700
148
38
59
53
785


NCU07308
2206
96
23
42
37
720


NCU05858
3
97
17
16
16
16


NCU01013
260
183
337
177
187
311


NCU06189
468
38
76
42
41
308


NCU05165
100
247
517
87
253
213


NCU04865
22
218
46
72
108
72


NCU05011
12
17
45
37
41
24


NCU00762
11
11
39334
33
26
292


NCU00836
10
9
9725
21
11
58


NCU01050
6
0.01
27848
3
0.01
9


NCU02240
8
48
19889
63
50
180


NCU02344
53
16
278
13
9
40


NCU02916
24
33
9996
39
57
79


NCU03328
7
162
7020
170
187
47


NCU04854
16
41
2195
44
46
61


NCU05057
11
31
12125
38
40
91


NCU05121
8
35
496
25
25
19


NCU07190
39
98
54230
101
383
1977


NCU07340
32
118
104476
170
152
274


NCU07760
28
19
174
32
27
80


NCU07898
3
15
7268
26
25
59


NCU08760
16
25
24669
31
19
219


NCU09680
41
99
62514
130
126
70


NCU03322
1
73
1360
641
782
1879


NCU07362
82
541
202
279
259
380


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


NCU12093
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


NCU10063
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


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


NCU10045
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


NCU04385
1604
43
14
18
18
1035


NCU02969
81
45
10
12
14
34


NCU08164
89
193
685
340
367
765


NCU00891
98
330
6350
7725
8502
22723


NCU08384
56
72
22043
28279
28261
76835


NCU08272
141
380
170
424
261
315


NCU07619
8
31
14
19
20
10


NCU05304
2192
188
412
175
219
837


NCU01510
15
772
319
623
681
124


NCU05768
6
39
0.01
2
0.01
0.01


NCU07154
12
158
65
101
93
23


NCU01998
241
46
107
96
84
246


NCU08457
200
210102
18274
13320
20799
1223


NCU06386
87
127
300
160
155
175


NCU09425
40
740
3087
1228
1964
538


NCU02478
152
16
57
32
39
39


NCU09175
2497
5190
10237
7817
8879
6804


NCU01689
1517
418
558
256
402
1214


NCU11721
233
209
367
173
156
242


NCU02396
131
66
27
59
33
24


NCU07481
76
25
62
43
46
121


NCU03137
271
112
308
146
138
153


NCU02500
29
1042
2415
0.01
0.01
732


NCU00565
498
254
92
217
155
206


NCU02705
245
54
26
117
61
37


NCU05225
57
312
115
176
161
170


NCU08326
204
119
62
220
91
73


NCU00326
465
430
1721
260
639
916


NCU08691
3
47
13
15
12
6


NCU09043
30
3328
1226
1683
1701
602


NCU07432
170
180
544
326
270
95


NCU05841
356
46
484
149
126
441


NCU02361
146
204
119
359
203
747


NCU10051
2833
301
12598
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1474
2773


NCU04720
1382
231
1993
748
1018
2367


NCU04698
349
178
419
238
318
176


NCU00177
2560
43
100
35
40
710


NCU01786
1012
45
117
32
53
560


NCU03117
6482
42
201
21
20
1298


NCU05254
229
17
33
11
16
106


NCU03963
1528
15
172
6
23
514


NCU09659
685
134
70
472
193
640


NCU03488
1689
1939
508
917
875
1054


NCU02657
11661
70
205
80
83
1464


NCU05855
13
20
315
42
49
41


NCU08044
385
420
1387
710
381
566


NCU09283
83
337
812
244
319
267


NCU11243
2
190
406
123
116
43


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
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485


NCU09553
201
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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


NCU02118
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
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NCU03319
940
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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
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1068
1948


NCU00169
486
315
1214
404
395
655


NCU02681
691
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1150
435
391
519


NCU06333
488
307
1502
340
339
589


NCU01146
1459
761
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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
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516


NCU08038
15
85
261
125
146
69


NCU02729
621
4
16
3
4
111


NCU03364
209
744
1586
596
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NCU03817
66
135
46
62
62
58


NCU06111
350
164
87
70
57
172


NCU08115
47
48
396
97
185
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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
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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
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2577
1577


NCU08561
24
410
62
316
152
28


NCU09287
8
3578
2172
6710
5148
1987


NCU00801
29
262
17161
861
1533
340


NCU00809
43
85
194
163
131
499


NCU07668
33
38
159
86
81
28


NCU05089
38
72
9
21
19
14


NCU08152
0.01
159
22
64
53
0.01


NCU01633
3273
118
394
1728
1321
36


NCU04537
12
15
35
118
84
339


NCU05853
17
1550
19630
5956
16364
2012


NCU08114
25
508
45944
13300
29186
11873


NCU00023
459
594
1250
411
450
554


NCU02009
295
443
4284
707
715
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NCU07068
7
2473
873
1574
1697
233


NCU03305
583
367
672
475
434
701


NCU08225
707
168
73
106
104
718


NCU08147
113
3406
1284
2158
2101
814


NCU06366
338
451
202
213
235
215


NCU05585
10
1426
433
802
647
307


NCU06138
11
167
3757
2567
2713
5610


NCU05591
59
94
312
270
403
105


NCU06032
84
137
334
117
120
135


NCU09098
40
72
196
60
69
57


NCU10009
103
37
84
42
51
102


NCU00290
2
172
38
140
78
31


NCU09580
7
40
12
45
31
14


NCU00803
22
157
57
77
76
17


NCU04374
16
195
37
62
67
49


NCU08425
14
364
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105
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10


NCU04097
1105
99
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151
99
83


NCU05079
364
981
1440
6283
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226


NCU07546
89
70
231
157
189
87


NCU08148
738
193
420
1416
611
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NCU03107
109
354
95
75
82
43


NCU00586
17
547
97
400
333
11


NCU00716
15
167
49
228
62
0.01


NCU00025
166
1481
553
924
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246


NCU00848
41
261
9
11
7
23


NCU00449
3
7
1235
8
12
36


NCU00849
4
66
9
6
0.01
0.01


NCU01058
6
14
73
12
30
0.01


NCU01076
0.01
3
784
0.01
0.01
0.01


NCU01196
60
55
88
41
30
127


NCU01978
15
21
47
17
24
21


NCU02138
113
158
462
69
59
55


NCU03083
677
273
890
355
347
593


NCU03982
2821
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5011


NCU04948
1
2
18
3
5
0.01


NCU05230
101
23
84
24
27
67


NCU05863
0.01
21
563
12
69
0.01


NCU05864
23
64
14268
143
96
212


NCU06152
4
45
76
23
35
37


NCU06607
35
21
794
31
26
20


NCU08756
4
10
120
10
46
28


NCU08790
1
10
389
16
44
103


NCU09295
321
86
292
115
131
348


NCU09524
3
14
2814
29
10
11


NCU11268
26
140
471
125
149
41


NCU11542
10
76
261
84
78
31


NCU11753
32
37
163
21
20
532


NCU00175
13
1227
188
932
575
83


NCU00250
9
234
67
187
144
0.01


NCU00322
174
566
235
882
592
55


NCU00695
55
1158
939
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2217
485


NCU07311
144
4941
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3903
3941
1195


NCU08171
509
1381
376
852
648
1136


NCU08521
0.01
20
9
14
15
0.01


NCU10507
3
251
91
182
143
58


NCU07143
11
86
13931
645
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NCU07222
48
2
147
42
22
114


NCU08371
54
176
706
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NCU09506
369
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6
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NCU04106
388
319
61
107
98
155


NCU06526
7
708
145
222
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35


NCU09196
4
94
42
51
60
10


NCU11466
2
221
92
133
152
14


NCU11957
0.01
19
7
14
7
0.01


NCU00995
2433
398
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3520
1448
924


NCU01720
931
61
178
270
226
760


NCU03293
12057
118
359
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656
5218


NCU04169
3
20
2545
5669
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803


NCU04170
11
35
203
466
412
40


NCU04467
63
56
320
323
235
71


NCU04932
197
136
873
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NCU04998
5
5
27
22
18
0.01


NCU05134
653
65
1366
826
1673
2090


NCU05350
1
37
315
191
286
447


NCU05829
294
22
65
55
45
139


NCU05852
4
67
200
161
219
35


NCU05908
7
20
44
67
27
138


NCU06143
16
147
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7304


NCU06983
564
103
620
1152
1130
1537


NCU06991
97
22
99
97
125
246


NCU08635
0.01
29
61
101
83
0.01


NCU09046
0.01
6
22
54
36
8


NCU09172
0.01
0.01
9
1
1
0.01


NCU09424
1
13
49
34
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20


NCU09498
340
67
252
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417
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NCU09823
28
101
350
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NCU09848
215
15
76
53
51
32


NCU10014
419
93
997
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NCU10039
8
56
888
376
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NCU10687
258
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165
219
241
958


NCU00561
23
601
165
233
249
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NCU00859
53
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184
112
162
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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


NCU12011
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


NCU01148
28
27
106
37
36
36


NCU01944
58
20
441
46
47
43


NCU01970
96
1490
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761
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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
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NCU03152
317
89
325
103
95
180


NCU03329
91
170
1134
326
293
135


NCU03433
17
8
39
10
10
18


NCU04127
2828
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3165
993
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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


NCU05170
318
205
652
343
208
463


NCU05569
128
74
147
37
42
61


NCU05574
22
59
699
46
156
328


NCU05846
43
120
3033
165
521
251


NCU05848
1
0.01
24
0.01
3
0.01


NCU05854
12
49
227
59
67
33


NCU06214
1375
143
262
90
107
327


NCU06312
5
34
62
17
27
12


NCU06704
981
347
1914
362
466
544


NCU07207
0.01
18
44
7
9
0.01


NCU07336
39
15
119
20
21
22


NCU07339
0.01
10
204
14
9
0.01


NCU07453
53
204
868
205
186
1016


NCU07897
6
16
123
22
20
12


NCU07979
7
31
196
42
48
17


NCU08043
0.01
13
42
8
5
0.01


NCU08113
4
4
266
7
32
50


NCU08117
75
35
174
35
56
82


NCU08379
1330
786
2359
903
1094
1579


NCU08624
11
26
342
35
31
14


NCU08784
8
61
303
48
63
32


NCU09003
201
69
133
75
90
180


NCU09426
0.01
29
103
35
50
6


NCU09479
11
21
38
21
19
12


NCU09522
87
46
203
54
51
55


NCU09523
26
27
207
51
34
11


NCU09689
8
105
5646
112
1811
1256


NCU10521
63
103
462
88
108
417


NCU11118
468
125
522
103
150
123


NCU11278
8
65
612
51
49
54


NCU11327
531
538
995
523
510
748


NCU11397
3483
48
161
56
59
648


NCU11690
26
60
158
46
54
27


NCU11722
471
224
571
251
200
327


NCU11862
13
96
273
69
66
52


NCU00247
27
149
82
225
136
74


NCU01347
98
168
116
220
143
38


NCU01598
4
54
17
40
27
0.01


NCU03761
105
144
53
210
77
27


NCU04635
109
863
439
1494
713
76


NCU04667
9
234
101
338
293
23


NCU05058
5
7
0.01
9
7
0.01


NCU05128
6
89
50
267
221
18


NCU06265
49
3080
1086
2116
2448
689


NCU06615
125
1123
359
1086
594
209


NCU06895
1
2463
677
5512
4070
38


NCU07233
23
153
120
271
172
38


NCU07423
113
92
53
124
77
41


NCU07424
12
190
94
169
127
32


NCU07895
105
920
343
1438
853
84


NCU08418
69
561
277
927
642
332


NCU08557
0.01
41
12
27
26
22


NCU08712
30
52
21
58
35
18


NCU09060
0.01
8
0.01
5
0.01
0.01


NCU09231
17
117
92
163
164
10


NCU09685
0.01
60
25
69
31
0.01


NCU09958
58
489
205
986
474
47


NCU10276
480
621
390
3406
2011
359


NCU11697
34
157
89
231
154
39


NCU11944
23
112
58
104
106
0.01


NCU12051
1
175
69
129
123
14


NCU12128
4
105
38
75
83
10


NCU12145
1
25
16
45
34
2


NCU00289
187
105
358
233
174
281


NCU00496
14
15
53
29
30
13


NCU00763
0.01
0.01
156
1
1
0.01


NCU01386
111
69
330
227
130
89


NCU02485
16
30
267
70
117
291


NCU02882
110
16
244
50
64
110


NCU04618
30
35
171
56
80
18


NCU04871
0.01
1
20
5
3
2


NCU04904
336
572
1657
872
880
731


NCU05351
20
14
61
45
26
0.01


NCU05501
12
55
470
151
120
128


NCU05906
9
207
501
304
274
340


NCU06373
9
42
228
92
82
12


NCU07270
11
19
62
26
33
49


NCU08116
2
3
15
6
8
0.01


NCU08397
12
224
1466
1969
617
81


NCU08748
77
71
188
101
116
186


NCU08867
87
17
168
47
42
34


NCU09176
10
63
573
152
198
194


NCU11769
8
6
23
9
12
8


NCU11828
912
76
895
237
210
99


NCU11905
2
3
26
11
8
0.01


NCU00011
0.01
21
6
11
10
0.01


NCU00397
259
1679
656
890
836
616


NCU00510
3
60
20
30
28
0.01


NCU00935
1326
1592
327
535
546
924


NCU01880
4
52
17
28
31
0.01


NCU02080
318
663
153
347
248
43


NCU02130
35
552
169
311
337
71


NCU02163
10
66
18
33
29
7


NCU02365
0.01
46
11
16
20
0.01


NCU03157
21
128
41
62
67
42


NCU03352
221
157
70
98
97
70


NCU03398
13
502
241
284
376
67


NCU03570
2
49
9
19
23
0.01


NCU04282
4
180
40
83
71
23


NCU04342
279
836
312
553
295
444


NCU04360
91
404
125
232
181
91


NCU04525
157
394
88
174
167
369


NCU04866
26
374
36
149
105
65


NCU05784
8
201
37
72
75
34


NCU05951
4
231
51
89
85
43


NCU05976
3
15
3
7
4
0.01


NCU06156
4
272
137
199
194
47


NCU06986
1777
5683
1405
2505
2588
2099


NCU07126
143
143
68
94
79
49


NCU07593
0.01
19
3
9
5
0.01


NCU07718
0.01
1234
102
379
310
9


NCU08224
482
1740
423
782
812
3564


NCU08469
3
864
221
427
452
263


NCU08726
34
225
72
128
124
46


NCU09049
1
115
16
36
31
0.01


NCU09115
89
1644
571
771
869
478


NCU09883
74
551
156
324
288
133


NCU10658
102
303
113
186
169
129


NCU10770
7
21
4
8
8
0.01


NCU11294
20
158
32
92
51
101


NCU00304
297
480
1160
1340
1020
820


NCU00798
1356
182
928
1074
1075
1877


NCU01136
81
36
80
94
95
200


NCU01430
8
92
670
291
316
1699


NCU03791
4480
221
524
572
481
1376


NCU04167
5
19
58
195
131
21


NCU04400
1
78
1893
963
1385
2507


NCU04557
1
2
27
15
13
6


NCU04879
10
0.01
11
56
27
0.01


NCU04910
1554
3082
8104
6702
5873
9988


NCU04928
16
3
22
18
10
0.01


NCU05068
39
39
2321
1095
1569
121


NCU05755
52
123
589
625
629
552


NCU05826
791
25
177
174
157
630


NCU05832
806
544
2806
1841
1558
3059


NCU05875
4
26
53
77
69
18


NCU05909
3
1
32
16
11
14


NCU06181
85
37
108
88
99
79


NCU06235
326
34
105
115
78
111


NCU06387
77
42
367
199
234
116


NCU07235
4
0.01
8
26
20
12


NCU07510
0.01
0.01
129
129
176
233


NCU07572
59
63
124
243
172
87


NCU07997
161
299
781
976
835
547


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


NCU10697
18
20
154
94
129
15


NCU11095
7
8
32
23
24
9


NCU11291
11
9
58
32
29
10


NCU11689
5
3
28
105
72
15


NCU11801
4
5
20
22
21
6


NCU11932
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


NCU01109
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


NCU03011
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


NCU08700
28
116
51
45
48
80


NCU08775
12
197
60
54
48
13


NCU09272
16
394
68
68
78
25


NCU09273
45
800
106
76
92
53


NCU09274
31
1352
109
93
117
36


NCU09335
15
448
133
151
213
50


NCU09342
3
18
2
3
2
0.01


NCU09714
10
256
119
136
130
82


NCU09782
113
736
273
286
384
124


NCU10062
305
169
71
81
91
86


NCU10301
7
103
51
65
42
36


NCU11565
29
89
64
56
60
23


NCU11774
1744
31112
14608
17568
17970
5816


NCU11881
72
587
201
227
263
133


NCU11974
603
8076
1100
1707
1525
288


NCU11989
36
812
234
272
343
76


NCU12012
0.01
7
0.01
0.01
0.01
0.01


NCU12014
0.01
6
0.01
0.01
0.01
0.01


NCU12015
0.01
6
0.01
0.01
0.01
0.01
















TABLE 1D







Differential expression (DE) patterns between


various mutant strains and growth conditions*













Avicel v
Avicel v
Avicel v
Sucrose v
Avicel v



No Car-
Δclr-1
Δclr-2
No Car-
Sucrose


Gene
bon DE
DE
DE
bon 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


NCU03648
cuffdiff
yes
yes
yes
yes


NCU03913
cuffdiff
yes
yes
yes
cuffdiff


NCU05499
cuffdiff
yes
yes
yes
yes


NCU05537
cuffdiff
yes
yes
yes
yes


NCU05977
yes
yes
yes
yes
yes


NCU06448
cuffdiff
yes
cuffdiff
cuffdiff


NCU06543
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU07153
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU08216
cuffdiff
yes
yes
cuffdiff
yes


NCU09116
yes
yes
yes
cuffdiff
yes


NCU09266
cuffdiff
yes
cuffdiff
yes
yes


NCU09864
cuffdiff
yes
yes
yes
yes


NCU11195
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU01830
cuffdiff
cuffdiff
yes
yes
yes


NCU02126
yes
yes
yes
yes
yes


NCU01744
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU03748
yes
cuffdiff
cuffdiff
yes
yes


NCU06625
yes
cuffdiff
cuffdiff
yes


NCU04130
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU10110
yes
cuffdiff
cuffdiff
yes
yes


NCU03861
yes


yes


NCU07623
yes


yes
yes


NCU01427
yes

cuffdiff
yes
yes


NCU03651
yes
cuffdiff
yes
yes
cuffdiff


NCU02579
yes
cuffdiff
cuffdiff
yes
yes


NCU07307
yes


yes
yes


NCU07308
yes


yes
yes


NCU05858
yes


yes
yes


NCU01013
yes
yes
yes
cuffdiff
cuffdiff


NCU06189
yes
cuffdiff
cuffdiff
yes
yes


NCU05165
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU04865
yes

cuffdiff
yes
cuffdiff


NCU05011
yes



yes


NCU00762
yes
yes
yes

yes


NCU00836
yes
yes
yes

yes


NCU01050
yes
yes
yes
cuffdiff
yes


NCU02240
yes
yes
yes
yes
yes


NCU02344
yes
yes
yes
yes
cuffdiff


NCU02916
yes
yes
yes

yes


NCU03328
yes
yes
yes
yes
yes


NCU04854
yes
yes
yes
yes
yes


NCU05057
yes
yes
yes
yes
yes


NCU05121
yes
yes
yes
yes
yes


NCU07190
yes
yes
yes
cuffdiff
yes


NCU07340
yes
yes
yes
cuffdiff
yes


NCU07760
yes
yes
yes
yes
cuffdiff


NCU07898
yes
yes
yes
yes
yes


NCU08760
yes
yes
yes

yes


NCU09680
yes
yes
yes

yes


NCU03322
yes
cuffdiff
cuffdiff
yes
yes


NCU07362
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU03813
yes
cuffdiff
cuffdiff
yes
yes


NCU04539
yes


yes
yes


NCU08687
yes
yes
yes
cuffdiff
yes


NCU05133
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU09705
yes
cuffdiff
cuffdiff
yes
yes


NCU07277
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU04797
yes
yes
cuffdiff
yes
cuffdiff


NCU00575
yes
cuffdiff
cuffdiff
yes


NCU04401
yes
cuffdiff
cuffdiff
yes
yes


NCU02855
yes
yes
yes

yes


NCU05924
yes
yes
yes

yes


NCU05955
yes
yes
yes
yes
yes


NCU07326
yes
yes
yes
yes
yes


NCU09775
yes
cuffdiff
yes
cuffdiff
yes


NCU04997
yes
yes
yes

yes


NCU01900
yes
cuffdiff
cuffdiff
yes
yes


NCU02343
yes
cuffdiff
cuffdiff
yes
yes


NCU07225
yes
yes
yes
cuffdiff
yes


NCU08087
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU08189
yes

cuffdiff
yes
yes


NCU09652
yes

cuffdiff
yes
yes


NCU06881
cuffdiff
yes
cuffdiff

cuffdiff


NCU01853
cuffdiff
yes
yes
yes
yes


NCU02287
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU02894
yes
cuffdiff
cuffdiff
cuffdiff


NCU07263
yes
yes
cuffdiff

yes


NCU08924
yes
cuffdiff
cuffdiff
yes
yes


NCU09692
yes
cuffdiff
cuffdiff
yes
yes


NCU04796
yes
cuffdiff
cuffdiff
yes
yes


NCU09732
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU07719
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU12093
yes
yes
cuffdiff
yes
yes


NCU05818
yes


yes
yes


NCU04078
yes
yes
yes
yes
yes


NCU07617
yes


yes
cuffdiff


NCU08398
yes
yes
yes
yes
yes


NCU10683
cuffdiff
cuffdiff
yes
yes
yes


NCU10063
yes


yes
cuffdiff


NCU04933
yes
cuffdiff
cuffdiff

cuffdiff


NCU00890
yes
yes
yes
yes
yes


NCU04623
yes
yes
cuffdiff
yes
yes


NCU04952
yes
yes
yes
yes
yes


NCU05956
cuffdiff
yes
cuffdiff
yes
yes


NCU07487
yes
yes
yes

yes


NCU08755
yes
yes
cuffdiff
yes
yes


NCU00130
yes
yes
yes
yes
yes


NCU00709
yes
cuffdiff
cuffdiff

yes


NCU04168
yes
yes
cuffdiff
yes
yes


NCU09904
yes
cuffdiff


yes


NCU09923
yes
cuffdiff
cuffdiff
yes
yes


NCU03098
yes

cuffdiff
yes
yes


NCU09028
yes


yes
cuffdiff


NCU09281
yes
cuffdiff
cuffdiff
yes
yes


NCU10107
yes
cuffdiff
cuffdiff
yes
yes


NCU00206
yes
yes
yes

yes


NCU00710
yes
yes
yes

yes


NCU01059
yes
yes
cuffdiff

yes


NCU03181
yes
yes
yes
yes
yes


NCU04494
yes
yes
yes

yes


NCU05598
yes
yes
yes
cuffdiff
cuffdiff


NCU05751
yes
yes
yes

yes


NCU08176
yes
yes
yes

yes


NCU08746
yes
yes
yes
yes
yes


NCU08785
yes
yes
yes
yes
yes


NCU09445
yes
yes
yes
cuffdiff
yes


NCU09491
yes
yes
yes
yes
yes


NCU09582
yes
yes
yes

yes


NCU09764
yes
yes
yes
yes
yes


NCU09774
yes
yes
yes

yes


NCU09976
yes
yes
yes

yes


NCU10045
yes
yes
yes
cuffdiff
yes


NCU11068
yes
yes
yes
yes
yes


NCU11198
yes
yes
yes

yes


NCU02904
yes
cuffdiff
cuffdiff
yes


NCU04870
yes
yes
yes

yes


NCU05159
yes
yes
yes
yes
yes


NCU09518
yes
cuffdiff
cuffdiff
yes
yes


NCU09664
yes
yes
yes

yes


NCU09924
yes
yes
yes
yes
yes


NCU03158
yes


cuffdiff
cuffdiff


NCU07067
yes
cuffdiff
cuffdiff
yes
yes


NCU01353
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU07269
yes


yes
yes


NCU06023
yes


yes


NCU06025
yes


cuffdiff


NCU00761
yes
yes
yes

yes


NCU06650
cuffdiff
cuffdiff
yes
yes
yes


NCU09416
yes
yes
yes
cuffdiff
yes


NCU00292
yes

cuffdiff
yes
yes


NCU03903
yes
cuffdiff
cuffdiff
yes
yes


NCU04475
yes
cuffdiff
cuffdiff
yes
yes


NCU06364
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU09575
yes


yes
yes


NCU04230
yes
yes
cuffdiff
yes
cuffdiff


NCU02366
yes


yes
cuffdiff


NCU04280
yes


yes
yes


NCU04385
yes


yes
yes


NCU02969
yes


cuffdiff
yes


NCU08164
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU00891
yes

cuffdiff
cuffdiff
yes


NCU08384
yes
cuffdiff


yes


NCU08272
cuffdiff
yes
cuffdiff
cuffdiff


NCU07619
yes


yes


NCU05304
yes
yes
cuffdiff
yes
yes


NCU01510
yes
cuffdiff
cuffdiff
yes
yes


NCU05768
yes
cuffdiff

yes
cuffdiff


NCU07154
yes
cuffdiff
cuffdiff
yes
yes


NCU01998
yes

cuffdiff
yes
cuffdiff


NCU08457
yes
cuffdiff
cuffdiff
yes
yes


NCU06386
yes
cuffdiff
cuffdiff

cuffdiff


NCU09425
yes
cuffdiff
cuffdiff
yes
yes


NCU02478
yes
cuffdiff

yes
cuffdiff


NCU09175
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU01689
cuffdiff
yes
cuffdiff
yes
yes


NCU11721
cuffdiff
yes
yes
cuffdiff
cuffdiff


NCU02396
yes
cuffdiff

cuffdiff
yes


NCU07481
yes


yes


NCU03137
yes
cuffdiff
cuffdiff
cuffdiff


NCU02500
cuffdiff
yes
yes
yes
yes


NCU00565
yes
yes
cuffdiff
cuffdiff
yes


NCU02705
cuffdiff
yes
yes
yes
yes


NCU05225
yes


cuffdiff
cuffdiff


NCU08326
cuffdiff
yes

cuffdiff
cuffdiff


NCU00326
yes
yes
yes
cuffdiff
yes


NCU08691
yes


yes
yes


NCU09043
yes
cuffdiff
cuffdiff
yes
yes


NCU07432
yes
cuffdiff
cuffdiff

cuffdiff


NCU05841
yes
cuffdiff
yes
yes
cuffdiff


NCU02361
cuffdiff
yes
cuffdiff


NCU10051
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU04720
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU04698
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU00177
cuffdiff
yes
cuffdiff
yes
yes


NCU01786
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU03117
cuffdiff
yes
yes
yes
yes


NCU05254
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU03963
yes
yes
yes
yes
yes


NCU09659
cuffdiff
yes
cuffdiff
yes
yes


NCU03488
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU02657
yes
cuffdiff
cuffdiff
yes
yes


NCU05855
yes
yes
yes

yes


NCU08044
yes
cuffdiff
yes

yes


NCU09283
cuffdiff
yes
cuffdiff
yes
yes


NCU11243
cuffdiff
yes
yes
yes
yes


NCU01378
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU01861
yes
yes
yes
yes
yes


NCU04583
cuffdiff
yes
yes
cuffdiff
cuffdiff


NCU06616
yes
yes
cuffdiff
yes
yes


NCU07325
yes
cuffdiff
cuffdiff
yes
yes


NCU08771
yes
yes
cuffdiff
yes
yes


NCU09553
cuffdiff
yes
cuffdiff
cuffdiff


NCU10055
cuffdiff
yes
yes
yes
yes


NCU11289
yes


yes


NCU08750
yes
yes
cuffdiff
yes
yes


NCU08752
yes
yes
cuffdiff
yes
yes


NCU03049
yes


yes
yes


NCU05653
yes



yes


NCU07133
yes
cuffdiff
cuffdiff
yes
yes


NCU08925
yes
cuffdiff

yes


NCU09865
yes


yes


NCU11365
yes
cuffdiff

cuffdiff


NCU07055
yes
cuffdiff
cuffdiff
yes
yes


NCU07224
yes
yes
yes

yes


NCU01061
yes


yes


NCU03566
yes


yes
cuffdiff


NCU04260
yes

cuffdiff
yes
yes


NCU05094
yes

cuffdiff
yes
yes


NCU05986
yes


yes
cuffdiff


NCU06153
yes
cuffdiff
cuffdiff
yes
yes


NCU09674
yes


cuffdiff
cuffdiff


NCU11241
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU03013
yes
cuffdiff
cuffdiff
yes
yes


NCU05319
yes
cuffdiff
cuffdiff
yes


NCU04430
cuffdiff
yes
yes
yes
yes


NCU02059
yes
yes
yes
yes
cuffdiff


NCU00831
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU06055
yes
cuffdiff
cuffdiff

cuffdiff


NCU00263
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU07200
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU09992
cuffdiff
yes
cuffdiff
yes
yes


NCU09265
yes
yes
yes
cuffdiff
yes


NCU00813
yes
yes
yes
cuffdiff
yes


NCU02455
yes
cuffdiff
cuffdiff

yes


NCU09223
yes
yes
yes
cuffdiff
yes


NCU09485
yes
yes
yes
cuffdiff
cuffdiff


NCU01648
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU10497
yes
yes
cuffdiff
cuffdiff
cuffdiff


NCU00669
yes
yes
cuffdiff
cuffdiff
cuffdiff


NCU02118
yes
cuffdiff
cuffdiff
cuffdiff


NCU10762
yes
cuffdiff
cuffdiff
yes


NCU00244
yes


cuffdiff
cuffdiff


NCU01068
yes
cuffdiff
cuffdiff
yes
yes


NCU03319
yes
cuffdiff
cuffdiff

yes


NCU08761
yes
yes
cuffdiff
cuffdiff
cuffdiff


NCU01279
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU03819
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU08607
yes
cuffdiff
cuffdiff

cuffdiff


NCU09195
yes
cuffdiff
cuffdiff
cuffdiff


NCU07736
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU01290
cuffdiff
yes
cuffdiff
yes
yes


NCU03396
cuffdiff
yes
cuffdiff
yes
yes


NCU09521
cuffdiff
yes
yes
yes
yes


NCU03897
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU07746
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU08897
yes
yes
yes
cuffdiff
cuffdiff


NCU00169
yes
yes
yes
cuffdiff
cuffdiff


NCU02681
yes
cuffdiff
yes
cuffdiff
cuffdiff


NCU06333
yes
yes
yes
cuffdiff
yes


NCU01146
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU00931
yes


yes
yes


NCU07008
yes


yes
cuffdiff


NCU03295
cuffdiff
yes
cuffdiff
yes
yes


NCU07737
yes
yes
cuffdiff
cuffdiff
cuffdiff


NCU08038
yes
cuffdiff
cuffdiff
yes
yes


NCU02729
cuffdiff
yes
cuffdiff
yes
yes


NCU03364
cuffdiff
yes
cuffdiff
yes
yes


NCU03817
yes


cuffdiff


NCU06111
yes


cuffdiff
yes


NCU08115
yes
yes
cuffdiff

yes


NCU06931
cuffdiff
yes
cuffdiff
yes
yes


NCU04077
yes
cuffdiff
cuffdiff
yes
yes


NCU01862
yes
cuffdiff
cuffdiff
yes
yes


NCU02795
yes


yes


NCU00812
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU01856
yes
yes
yes
cuffdiff
cuffdiff


NCU03725
yes
yes
yes
yes
cuffdiff


NCU06971
yes
yes
yes
yes
yes


NCU07705
yes
yes
cuffdiff
yes
yes


NCU08042
yes
yes
yes

yes


NCU03643
yes
yes
yes
yes
yes


NCU03043
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU05767
yes


yes
yes


NCU00316
cuffdiff
yes
cuffdiff
cuffdiff


NCU00721
cuffdiff
yes
yes
yes
cuffdiff


NCU07578
cuffdiff
yes
cuffdiff
yes
yes


NCU04435
yes


NCU05198
yes


cuffdiff
yes


NCU10721
yes
yes
yes

yes


NCU11342
yes
yes
yes
yes
yes


NCU00821
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU08561
yes
yes
cuffdiff
yes
cuffdiff


NCU09287
cuffdiff
yes
cuffdiff
yes
yes


NCU00801
yes
yes
yes
yes
yes


NCU00809
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU07668
yes
cuffdiff
cuffdiff

yes


NCU05089
yes


cuffdiff
yes


NCU08152
yes
cuffdiff
cuffdiff
yes
yes


NCU01633
cuffdiff
yes
cuffdiff
yes
yes


NCU04537
yes
cuffdiff
cuffdiff

yes


NCU05853
yes
cuffdiff
cuffdiff
yes
yes


NCU08114
yes
yes
cuffdiff
yes
yes


NCU00023
cuffdiff
yes
yes
cuffdiff
yes


NCU02009
yes
yes
yes
cuffdiff
yes


NCU07068
yes
cuffdiff
cuffdiff
yes
yes


NCU03305
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU08225
yes
cuffdiff
cuffdiff
yes
yes


NCU08147
yes
cuffdiff
cuffdiff
yes
yes


NCU06366
yes


cuffdiff
cuffdiff


NCU05585
yes
cuffdiff
cuffdiff
yes
yes


NCU06138
yes
cuffdiff
cuffdiff
yes
yes


NCU05591
yes
cuffdiff


yes


NCU06032
yes
yes
yes
cuffdiff
yes


NCU09098
yes
yes
yes

yes


NCU10009
yes
cuffdiff
cuffdiff
cuffdiff


NCU00290
yes
yes
cuffdiff
yes
yes


NCU09580
yes
yes

yes


NCU00803
yes


yes
yes


NCU04374
yes

cuffdiff
yes
cuffdiff


NCU08425
yes
yes
yes
yes
cuffdiff


NCU04097
cuffdiff
yes
cuffdiff
yes
yes


NCU05079
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU07546
yes
cuffdiff
cuffdiff

yes


NCU08148
cuffdiff
yes

yes
cuffdiff


NCU03107
yes


yes


NCU00586
yes
yes
yes
yes
cuffdiff


NCU00716
cuffdiff
yes

yes
cuffdiff


NCU00025
yes
cuffdiff
cuffdiff
yes
yes


NCU00848
yes


yes
yes


NCU00449
yes
yes
yes
cuffdiff
yes


NCU00849
yes

cuffdiff
yes


NCU01058
yes
yes
cuffdiff
yes
yes


NCU01076
yes
yes
yes
cuffdiff
yes


NCU01196
cuffdiff
cuffdiff
yes

cuffdiff


NCU01978
cuffdiff
yes
cuffdiff

yes


NCU02138
cuffdiff
yes
yes
cuffdiff
cuffdiff


NCU03083
yes
yes
yes
yes
cuffdiff


NCU03982
yes
yes
yes
cuffdiff
yes


NCU04948
yes
yes
cuffdiff
yes
yes


NCU05230
yes
yes
cuffdiff

yes


NCU05863
yes
yes
yes
yes
yes


NCU05864
yes
yes
yes
yes
yes


NCU06152
cuffdiff
yes
cuffdiff
yes
yes


NCU06607
yes
yes
yes
cuffdiff
yes


NCU08756
yes
yes
yes

yes


NCU08790
yes
yes
yes

yes


NCU09295
yes
yes
cuffdiff
yes


NCU09524
yes
yes
yes
yes
yes


NCU11268
yes
yes
cuffdiff
yes
yes


NCU11542
yes
yes
yes
yes
yes


NCU11753
cuffdiff
yes
yes

cuffdiff


NCU00175
yes
yes
yes
yes
yes


NCU00250
yes
yes
cuffdiff
yes
yes


NCU00322
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU00695
cuffdiff
yes
yes
yes
yes


NCU07311
yes
yes
yes
yes
yes


NCU08171
yes
yes
cuffdiff
cuffdiff
cuffdiff


NCU08521
yes


yes
cuffdiff


NCU10507
yes
cuffdiff
cuffdiff
yes
yes


NCU07143
yes
yes
yes
cuffdiff
yes


NCU07222
yes
yes
yes
yes
cuffdiff


NCU08371
yes
cuffdiff
cuffdiff
yes
yes


NCU09506
yes
cuffdiff
yes
yes
yes


NCU04106
yes
cuffdiff
cuffdiff

yes


NCU06526
yes
cuffdiff
cuffdiff
yes
yes


NCU09196
yes

cuffdiff
yes
yes


NCU11466
yes
cuffdiff
cuffdiff
yes
yes


NCU11957
yes


yes
cuffdiff


NCU00995
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU01720
yes


yes
yes


NCU03293
yes
cuffdiff
cuffdiff
yes
yes


NCU04169
yes
cuffdiff
cuffdiff
yes
yes


NCU04170
yes
cuffdiff
cuffdiff
yes
yes


NCU04467
yes

cuffdiff

yes


NCU04932
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU04998
yes



cuffdiff


NCU05134
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU05350
yes
cuffdiff

yes
yes


NCU05829
yes


yes
yes


NCU05852
yes


yes
yes


NCU05908
yes

cuffdiff

yes


NCU06143
yes
cuffdiff
cuffdiff
yes
yes


NCU06983
yes
cuffdiff
cuffdiff
yes


NCU06991
yes


yes


NCU08635
yes


yes
yes


NCU09046
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU09172
yes



yes


NCU09424
yes


yes
yes


NCU09498
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU09823
yes
yes
cuffdiff
cuffdiff
yes


NCU09848
yes


yes
cuffdiff


NCU10014
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU10039
yes
cuffdiff
cuffdiff
yes
yes


NCU10687
yes


yes
cuffdiff


NCU00561
yes
cuffdiff
cuffdiff
yes
yes


NCU00859
yes
cuffdiff

yes
yes


NCU02042
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU02164
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU04482
yes


yes


NCU04486
yes


yes
yes


NCU05236
yes


yes
yes


NCU05761
yes


yes


NCU05763
yes


yes


NCU06328
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU07948
yes


yes
yes


NCU08140
yes


yes


NCU08447
yes


yes
yes


NCU09734
yes


yes


NCU12011
yes


yes
cuffdiff


NCU00408
cuffdiff
yes
yes
yes
cuffdiff


NCU00633
yes
yes
cuffdiff
yes
cuffdiff


NCU00870
yes
yes
yes
cuffdiff
yes


NCU00871
yes
yes
yes
cuffdiff
yes


NCU00965
yes
yes
yes

yes


NCU01003
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU01049
yes
yes
yes

yes


NCU01077
yes
yes
yes

yes


NCU01148
yes
yes
yes

yes


NCU01944
yes
yes
yes
yes
yes


NCU01970
cuffdiff
yes
yes
yes
yes


NCU01983
yes
yes
yes
yes
yes


NCU02008
cuffdiff
yes
yes

yes


NCU02061
yes
yes
yes

cuffdiff


NCU02600
yes
yes
yes

yes


NCU02625
yes
yes
yes
cuffdiff
yes


NCU02720
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU02915
yes
yes
yes
yes
yes


NCU03152
yes
yes
yes
yes


NCU03329
yes
yes
yes
yes
yes


NCU03433
yes
yes
yes

cuffdiff


NCU04127
yes
yes
yes
cuffdiff
cuffdiff


NCU04522
yes
yes
yes
cuffdiff
yes


NCU04830
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU04905
yes
cuffdiff
yes

yes


NCU05056
yes
yes
yes

yes


NCU05170
yes
cuffdiff
yes

cuffdiff


NCU05569
cuffdiff
yes
yes
cuffdiff


NCU05574
yes
yes
yes
yes
yes


NCU05846
yes
yes
yes
yes
yes


NCU05848
yes
yes
yes
cuffdiff
yes


NCU05854
yes
cuffdiff
cuffdiff
yes
yes


NCU06214
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU06312
cuffdiff
yes
cuffdiff
yes
yes


NCU06704
yes
yes
yes
cuffdiff
cuffdiff


NCU07207
cuffdiff
yes
yes
yes
yes


NCU07336
yes
yes
yes

yes


NCU07339
yes
yes
yes
yes
yes


NCU07453
yes
yes
yes
yes
yes


NCU07897
yes
yes
yes
cuffdiff
yes


NCU07979
yes
yes
yes
yes
yes


NCU08043
yes
yes
yes
yes
yes


NCU08113
yes
yes
yes

yes


NCU08117
yes
yes
yes
cuffdiff
cuffdiff


NCU08379
yes
yes
yes
cuffdiff
cuffdiff


NCU08624
yes
yes
yes
cuffdiff
yes


NCU08784
yes
yes
cuffdiff
yes
yes


NCU09003
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU09426
yes
yes
cuffdiff
yes
yes


NCU09479
cuffdiff
cuffdiff
yes

yes


NCU09522
yes
yes
yes
cuffdiff
cuffdiff


NCU09523
yes
yes
yes

yes


NCU09689
yes
yes
yes
yes
yes


NCU10521
yes
yes
yes

cuffdiff


NCU11118
yes
yes
yes
cuffdiff


NCU11278
yes
yes
yes
yes
yes


NCU11327
cuffdiff
cuffdiff
yes

cuffdiff


NCU11397
yes
yes
yes
yes
yes


NCU11690
yes
yes
yes
cuffdiff
yes


NCU11722
yes
yes
yes
yes
cuffdiff


NCU11862
cuffdiff
yes
yes
yes
yes


NCU00247
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU01347
cuffdiff
yes
cuffdiff
cuffdiff


NCU01598
yes
yes

yes
yes


NCU03761
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU04635
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU04667
cuffdiff
yes
cuffdiff
yes
yes


NCU05058
yes
yes
yes

cuffdiff


NCU05128
cuffdiff
yes
yes
yes
yes


NCU06265
yes
cuffdiff
cuffdiff
yes
yes


NCU06615
yes
yes
cuffdiff
yes
yes


NCU06895
cuffdiff
yes
yes
yes
yes


NCU07233
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU07423
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU07424
yes
cuffdiff
cuffdiff
yes
yes


NCU07895
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU08418
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU08557
yes


yes
cuffdiff


NCU08712
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU09060
yes
yes

yes


NCU09231
cuffdiff
yes
yes
yes
yes


NCU09685
cuffdiff
yes

yes
yes


NCU09958
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU10276
cuffdiff
yes
yes
cuffdiff
cuffdiff


NCU11697
cuffdiff
yes
cuffdiff
yes
cuffdiff


NCU11944
yes
cuffdiff
yes
yes
yes


NCU12051
yes
cuffdiff
cuffdiff
yes
yes


NCU12128
yes
cuffdiff
cuffdiff
yes
yes


NCU12145
yes
yes
yes
yes
yes


NCU00289
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU00496
yes



yes


NCU00763
yes
yes
yes
cuffdiff
yes


NCU01386
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU02485
yes
cuffdiff
cuffdiff

yes


NCU02882
yes
yes
cuffdiff
yes
yes


NCU04618
yes
cuffdiff
cuffdiff

yes


NCU04871
yes
yes
yes

yes


NCU04904
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU05351
yes

cuffdiff

cuffdiff


NCU05501
yes
yes
yes
yes
yes


NCU05906
yes
cuffdiff
cuffdiff
yes
yes


NCU06373
yes
yes
yes
yes
yes


NCU07270
yes
cuffdiff
cuffdiff

yes


NCU08116
yes



yes


NCU08397
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU08748
yes
cuffdiff
cuffdiff

cuffdiff


NCU08867
yes
yes
yes
yes
cuffdiff


NCU09176
yes
cuffdiff
cuffdiff
yes
yes


NCU11769
yes



yes


NCU11828
yes
yes
yes
yes


NCU11905
yes

cuffdiff

yes


NCU00011
yes


yes
cuffdiff


NCU00397
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU00510
yes


yes
cuffdiff


NCU00935
yes
cuffdiff
cuffdiff

yes


NCU01880
yes


yes
yes


NCU02080
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU02130
yes
cuffdiff
cuffdiff
yes
yes


NCU02163
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU02365
yes


yes
cuffdiff


NCU03157
yes


yes
cuffdiff


NCU03352
yes
cuffdiff
cuffdiff

cuffdiff


NCU03398
yes
cuffdiff
cuffdiff
yes
yes


NCU03570
yes


yes


NCU04282
yes
cuffdiff
cuffdiff
yes
yes


NCU04342
yes
cuffdiff
cuffdiff
cuffdiff


NCU04360
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU04525
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU04866
yes
yes
cuffdiff
yes


NCU05784
yes
cuffdiff
cuffdiff
yes
yes


NCU05951
yes


yes
yes


NCU05976
yes


yes


NCU06156
yes
cuffdiff
cuffdiff
yes
yes


NCU06986
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU07126
yes
cuffdiff

cuffdiff
cuffdiff


NCU07593
yes


yes
cuffdiff


NCU07718
yes
yes
yes
yes
yes


NCU08224
yes
cuffdiff
cuffdiff
yes


NCU08469
yes
yes
yes
yes
yes


NCU08726
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU09049
yes


yes
yes


NCU09115
yes
cuffdiff
cuffdiff
yes
yes


NCU09883
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU10658
yes
cuffdiff
cuffdiff
yes


NCU10770
yes


cuffdiff


NCU11294
yes
cuffdiff

cuffdiff


NCU00304
yes
cuffdiff

cuffdiff
cuffdiff


NCU00798
yes


yes
cuffdiff


NCU01136
yes


yes


NCU01430
yes
cuffdiff
cuffdiff
yes
yes


NCU03791
yes


yes
yes


NCU04167
yes
yes
cuffdiff
yes
yes


NCU04400
yes
cuffdiff
cuffdiff
yes
yes


NCU04557
yes



yes


NCU04879
cuffdiff
yes

cuffdiff


NCU04910
yes

cuffdiff
cuffdiff
yes


NCU04928
yes


yes


NCU05068
yes
cuffdiff
cuffdiff

yes


NCU05755
yes
cuffdiff
cuffdiff
yes
yes


NCU05826
yes


yes
yes


NCU05832
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU05875
yes


yes
yes


NCU05909
yes

cuffdiff

yes


NCU06181
yes


yes


NCU06235
yes


yes
yes


NCU06387
yes
cuffdiff
cuffdiff
cuffdiff
yes


NCU07235
cuffdiff
yes
yes
cuffdiff


NCU07510
yes

cuffdiff

yes


NCU07572
cuffdiff
yes
cuffdiff

cuffdiff


NCU07997
yes
cuffdiff

cuffdiff
yes


NCU08383
yes


cuffdiff
yes


NCU08491
cuffdiff
yes
cuffdiff
yes
yes


NCU08634
yes


yes
yes


NCU09075
cuffdiff
yes

cuffdiff


NCU09415
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU09856
yes


yes
yes


NCU09874
cuffdiff
yes
cuffdiff
cuffdiff
cuffdiff


NCU09906
yes
cuffdiff
cuffdiff

cuffdiff


NCU10284
yes

cuffdiff
yes
cuffdiff


NCU10697
yes
cuffdiff


yes


NCU11095
yes



yes


NCU11291
yes
cuffdiff
cuffdiff

cuffdiff


NCU11689
cuffdiff
yes
yes

cuffdiff


NCU11801
yes



cuffdiff


NCU11932
yes
cuffdiff

yes
yes


NCU00365
yes


cuffdiff
cuffdiff


NCU00375
yes


yes
yes


NCU00755
yes


yes
yes


NCU01109
yes


yes
yes


NCU01292
yes


yes


NCU01551
yes


yes
cuffdiff


NCU01649
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU03011
yes


yes


NCU03417
yes
cuffdiff

yes


NCU04285
yes


yes


NCU04843
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU04851
yes


yes


NCU04861
yes


yes
yes


NCU04862
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU05006
yes


yes
cuffdiff


NCU05189
yes


yes


NCU05197
yes
cuffdiff
cuffdiff
cuffdiff
cuffdiff


NCU05477
yes


yes


NCU05762
yes


yes


NCU05764
yes


yes


NCU05766
yes


yes
yes


NCU05859
yes


yes


NCU05933
yes


yes
yes


NCU06334
yes


yes
yes


NCU07180
yes


yes
yes


NCU07363
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU08037
yes

cuffdiff
yes
yes


NCU08155
yes
cuffdiff

yes
cuffdiff


NCU08156
yes


yes
yes


NCU08170
yes


yes


NCU08455
yes
cuffdiff
cuffdiff
yes
yes


NCU08554
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU08622
yes


yes
yes


NCU08700
yes


yes
cuffdiff


NCU08775
yes


yes
yes


NCU09272
yes


yes
yes


NCU09273
yes


yes
cuffdiff


NCU09274
yes


yes
cuffdiff


NCU09335
yes

cuffdiff
yes
yes


NCU09342
yes


yes


NCU09714
yes
cuffdiff

yes
yes


NCU09782
yes

cuffdiff
yes
cuffdiff


NCU10062
yes


cuffdiff
cuffdiff


NCU10301
yes


yes
yes


NCU11565
yes


yes
cuffdiff


NCU11774
yes
cuffdiff
cuffdiff
yes
yes


NCU11881
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU11974
yes
cuffdiff
cuffdiff
yes
cuffdiff


NCU11989
yes
cuffdiff
cuffdiff
yes
yes


NCU12012
yes


yes


NCU12014
yes


yes


NCU12015
yes


yes
















TABLE 1E







Differential expression (DE) between


various conditions, continued*











Sucrose V Both
No Carbon v Both
Δclr-1 v


Gene
Mutants DE
Mutants DE
Δclr-2 DE





NCU00554
cuffdiff




NCU00944


NCU01195
cuffdiff
cuffdiff


NCU02785
cuffdiff


NCU02954
cuffdiff


NCU03131
cuffdiff


NCU04216
cuffdiff


NCU04298


NCU04837
cuffdiff


NCU05548
cuffdiff
cuffdiff


NCU07413
cuffdiff
cuffdiff


NCU10283


NCU00461
cuffdiff
cuffdiff
yes


NCU00591

cuffdiff
cuffdiff


NCU00680
cuffdiff

cuffdiff


NCU01402

yes


NCU02127
cuffdiff
cuffdiff
cuffdiff


NCU02704

cuffdiff
yes


NCU02727
cuffdiff
cuffdiff
yes


NCU02936

cuffdiff
yes


NCU03076
cuffdiff
cuffdiff
cuffdiff


NCU03415

cuffdiff
cuffdiff


NCU03648

cuffdiff
cuffdiff


NCU03913

cuffdiff
yes


NCU05499

cuffdiff
cuffdiff


NCU05537
cuffdiff
cuffdiff
cuffdiff


NCU05977

cuffdiff
cuffdiff


NCU06448
cuffdiff
cuffdiff
cuffdiff


NCU06543

cuffdiff
cuffdiff


NCU07153
cuffdiff
cuffdiff
cuffdiff


NCU08216
cuffdiff
cuffdiff
cuffdiff


NCU09116

cuffdiff
cuffdiff


NCU09266
cuffdiff
cuffdiff
cuffdiff


NCU09864
cuffdiff
cuffdiff
cuffdiff


NCU11195
cuffdiff
cuffdiff
cuffdiff


NCU01830

yes
cuffdiff


NCU02126

cuffdiff
cuffdiff


NCU01744
cuffdiff
cuffdiff
cuffdiff


NCU03748
cuffdiff
cuffdiff


NCU06625
cuffdiff
yes


NCU04130
cuffdiff
cuffdiff
yes


NCU10110

yes
cuffdiff


NCU03861

yes


NCU07623

yes


NCU01427
cuffdiff
yes


NCU03651
cuffdiff
cuffdiff
yes


NCU02579
cuffdiff
cuffdiff


NCU07307

yes


NCU07308
cuffdiff
yes


NCU05858

yes


NCU01013


NCU06189
cuffdiff


NCU05165
cuffdiff
cuffdiff
yes


NCU04865
cuffdiff
yes


NCU05011

yes


NCU00762
cuffdiff


NCU00836


NCU01050
cuffdiff
cuffdiff
cuffdiff


NCU02240


NCU02344


NCU02916


NCU03328


NCU04854


NCU05057


NCU05121


NCU07190

cuffdiff
yes


NCU07340
cuffdiff


NCU07760
yes


NCU07898
cuffdiff


NCU08760


NCU09680


NCU03322

yes
cuffdiff


NCU07362
cuffdiff
cuffdiff


NCU03813

cuffdiff
cuffdiff


NCU04539

cuffdiff


NCU08687
cuffdiff
cuffdiff


NCU05133
cuffdiff
yes
cuffdiff


NCU09705

yes
cuffdiff


NCU07277
cuffdiff
yes


NCU04797
cuffdiff
cuffdiff
yes


NCU00575
cuffdiff
cuffdiff


NCU04401

yes
cuffdiff


NCU02855

yes


NCU05924
cuffdiff


NCU05955


NCU07326


NCU09775


NCU04997


NCU01900

yes
cuffdiff


NCU02343

yes
cuffdiff


NCU07225
cuffdiff
yes
cuffdiff


NCU08087
yes
yes


NCU08189
cuffdiff
yes
cuffdiff


NCU09652

yes
cuffdiff


NCU06881

cuffdiff
cuffdiff


NCU01853


NCU02287

cuffdiff
cuffdiff


NCU02894

cuffdiff


NCU07263
cuffdiff

yes


NCU08924
cuffdiff
cuffdiff
cuffdiff


NCU09692
yes
cuffdiff


NCU04796

cuffdiff


NCU09732
cuffdiff
cuffdiff
yes


NCU07719

cuffdiff


NCU12093

cuffdiff


NCU05818

yes


NCU04078

cuffdiff
yes


NCU07617

yes


NCU08398
cuffdiff

yes


NCU10683


NCU10063

yes


NCU04933
cuffdiff
yes


NCU00890

cuffdiff
yes


NCU04623


cuffdiff


NCU04952

cuffdiff


NCU05956


NCU07487


NCU08755

cuffdiff
yes


NCU00130

yes
yes


NCU00709

yes


NCU04168

yes
cuffdiff


NCU09904
yes
yes


NCU09923

yes


NCU03098

yes


NCU09028

yes


NCU09281

yes


NCU10107

yes
cuffdiff


NCU00206


NCU00710

yes


NCU01059


yes


NCU03181


NCU04494


NCU05598
cuffdiff


NCU05751


NCU08176


NCU08746


yes


NCU08785
cuffdiff
yes


NCU09445

cuffdiff
cuffdiff


NCU09491


NCU09582

yes


NCU09764


NCU09774


NCU09976


NCU10045

yes


NCU11068

cuffdiff
yes


NCU11198


NCU02904
cuffdiff


NCU04870
cuffdiff
yes


NCU05159

yes
cuffdiff


NCU09518


NCU09664

cuffdiff


NCU09924

yes


NCU03158

cuffdiff


NCU07067

yes


NCU01353
cuffdiff
yes


NCU07269

yes


NCU06023

cuffdiff


NCU06025

yes


NCU00761


NCU06650
yes
yes
cuffdiff


NCU09416
cuffdiff
cuffdiff


NCU00292

yes
cuffdiff


NCU03903

yes


NCU04475

yes
cuffdiff


NCU06364

yes
cuffdiff


NCU09575

yes


NCU04230
cuffdiff
cuffdiff
yes


NCU02366
cuffdiff
yes


NCU04280
cuffdiff
yes


NCU04385
cuffdiff
yes


NCU02969

yes


NCU08164
cuffdiff
cuffdiff


NCU00891
cuffdiff
yes
cuffdiff


NCU08384

yes
cuffdiff


NCU08272


cuffdiff


NCU07619


NCU05304
cuffdiff


NCU01510
yes
cuffdiff


NCU05768

yes
cuffdiff


NCU07154

cuffdiff
cuffdiff


NCU01998
cuffdiff
cuffdiff


NCU08457
yes
yes
cuffdiff


NCU06386


NCU09425

cuffdiff
cuffdiff


NCU02478
cuffdiff
cuffdiff


NCU09175
cuffdiff
cuffdiff
cuffdiff


NCU01689

cuffdiff
cuffdiff


NCU11721
cuffdiff


NCU02396
cuffdiff

cuffdiff


NCU07481


NCU03137


NCU02500
yes
yes


NCU00565
cuffdiff
cuffdiff
cuffdiff


NCU02705
cuffdiff
cuffdiff
cuffdiff


NCU05225
cuffdiff
cuffdiff


NCU08326


yes


NCU00326
cuffdiff

yes


NCU08691

yes


NCU09043
yes
cuffdiff


NCU07432
cuffdiff
cuffdiff


NCU05841

yes


NCU02361
cuffdiff
cuffdiff
cuffdiff


NCU10051
cuffdiff
yes
yes


NCU04720
cuffdiff
yes
cuffdiff


NCU04698
cuffdiff
cuffdiff
cuffdiff


NCU00177
cuffdiff


NCU01786
cuffdiff


NCU03117
cuffdiff
cuffdiff


NCU05254
cuffdiff


NCU03963
cuffdiff

yes


NCU09659
cuffdiff
cuffdiff
yes


NCU03488
cuffdiff
yes


NCU02657
cuffdiff


NCU05855
cuffdiff
yes


NCU08044
cuffdiff
cuffdiff
cuffdiff


NCU09283
cuffdiff
cuffdiff
cuffdiff


NCU11243

cuffdiff


NCU01378
cuffdiff
cuffdiff


NCU01861
cuffdiff
cuffdiff
cuffdiff


NCU04583
cuffdiff
yes


NCU06616
cuffdiff
cuffdiff
yes


NCU07325

cuffdiff


NCU08771

cuffdiff
cuffdiff


NCU09553

cuffdiff
cuffdiff


NCU10055


NCU11289


NCU08750
cuffdiff
yes
cuffdiff


NCU08752

yes
yes


NCU03049

cuffdiff


NCU05653

cuffdiff


NCU07133

cuffdiff
cuffdiff


NCU08925

cuffdiff


NCU09865


NCU11365
cuffdiff
cuffdiff


NCU07055
cuffdiff
yes


NCU07224

yes


NCU01061
cuffdiff
yes


NCU03566

cuffdiff


NCU04260

yes


NCU05094

yes


NCU05986

cuffdiff


NCU06153

yes


NCU09674
cuffdiff
yes


NCU11241
cuffdiff
yes


NCU03013
cuffdiff
yes
cuffdiff


NCU05319
cuffdiff


NCU04430

yes
yes


NCU02059
yes
cuffdiff


NCU00831
cuffdiff
cuffdiff
cuffdiff


NCU06055

cuffdiff


NCU00263
cuffdiff
yes
yes


NCU07200
cuffdiff
yes
yes


NCU09992

yes
yes


NCU09265
cuffdiff
cuffdiff
cuffdiff


NCU00813
cuffdiff


NCU02455
cuffdiff
cuffdiff
cuffdiff


NCU09223
cuffdiff
cuffdiff


NCU09485

cuffdiff


NCU01648
cuffdiff
cuffdiff


NCU10497


cuffdiff


NCU00669


NCU02118


NCU10762


NCU00244
cuffdiff
cuffdiff


NCU01068
cuffdiff
cuffdiff


NCU03319
cuffdiff
cuffdiff


NCU08761


NCU01279
cuffdiff
cuffdiff


NCU03819

cuffdiff


NCU08607
cuffdiff
cuffdiff


NCU09195

cuffdiff


NCU07736
cuffdiff
yes
cuffdiff


NCU01290
cuffdiff


NCU03396
cuffdiff


NCU09521
cuffdiff


NCU03897


NCU07746
cuffdiff


NCU08897

cuffdiff


NCU00169

cuffdiff


NCU02681
cuffdiff


NCU06333


NCU01146

cuffdiff


NCU00931
cuffdiff


NCU07008

yes


NCU03295

cuffdiff
cuffdiff


NCU07737
cuffdiff
yes
cuffdiff


NCU08038

cuffdiff


NCU02729
cuffdiff


NCU03364

cuffdiff


NCU03817

yes


NCU06111
cuffdiff
yes


NCU08115

yes
cuffdiff


NCU06931


NCU04077
cuffdiff
cuffdiff


NCU01862

cuffdiff


NCU02795

cuffdiff


NCU00812
cuffdiff

cuffdiff


NCU01856
cuffdiff
cuffdiff


NCU03725
cuffdiff
cuffdiff


NCU06971


cuffdiff


NCU07705

cuffdiff
yes


NCU08042


yes


NCU03643


cuffdiff


NCU03043
cuffdiff
cuffdiff
cuffdiff


NCU05767

yes


NCU00316


cuffdiff


NCU00721
cuffdiff
yes


NCU07578
cuffdiff
cuffdiff


NCU04435


NCU05198

cuffdiff


NCU10721


NCU11342


NCU00821

cuffdiff
cuffdiff


NCU08561

cuffdiff
yes


NCU09287

cuffdiff
cuffdiff


NCU00801

yes
cuffdiff


NCU00809

cuffdiff


NCU07668

yes


NCU05089

yes


NCU08152

yes


NCU01633
cuffdiff
yes
cuffdiff


NCU04537

yes


NCU05853

yes
yes


NCU08114
cuffdiff
yes
yes


NCU00023

cuffdiff


NCU02009
yes
cuffdiff


NCU07068

cuffdiff


NCU03305

cuffdiff


NCU08225
cuffdiff
cuffdiff


NCU08147
yes
cuffdiff


NCU06366
cuffdiff
cuffdiff


NCU05585

cuffdiff
cuffdiff


NCU06138

yes
cuffdiff


NCU05591

yes
cuffdiff


NCU06032


NCU09098
cuffdiff


NCU10009


NCU00290

cuffdiff
cuffdiff


NCU09580


NCU00803

yes


NCU04374

yes


NCU08425

yes


NCU04097
cuffdiff

cuffdiff


NCU05079
cuffdiff
yes
cuffdiff


NCU07546

yes


NCU08148
cuffdiff
yes
yes


NCU03107

yes


NCU00586
cuffdiff
cuffdiff


NCU00716
cuffdiff

yes


NCU00025
cuffdiff
cuffdiff


NCU00848
cuffdiff
yes


NCU00449
cuffdiff
cuffdiff
cuffdiff


NCU00849

yes
yes


NCU01058


NCU01076


NCU01196


NCU01978


NCU02138
cuffdiff
yes


NCU03083

cuffdiff


NCU03982
cuffdiff
cuffdiff


NCU04948
cuffdiff


NCU05230


NCU05863

cuffdiff
yes


NCU05864

cuffdiff
cuffdiff


NCU06152


NCU06607


NCU08756

cuffdiff
yes


NCU08790
cuffdiff
cuffdiff
yes


NCU09295
cuffdiff
cuffdiff


NCU09524


yes


NCU11268


NCU11542


NCU11753
yes


NCU00175

cuffdiff
cuffdiff


NCU00250
yes
cuffdiff


NCU00322
cuffdiff
cuffdiff
cuffdiff


NCU00695
cuffdiff
cuffdiff
cuffdiff


NCU07311
cuffdiff
cuffdiff


NCU08171
yes
cuffdiff
cuffdiff


NCU08521


NCU10507

cuffdiff


NCU07143

yes
cuffdiff


NCU07222
cuffdiff
yes


NCU08371

cuffdiff


NCU09506


NCU04106
cuffdiff
yes


NCU06526

yes
cuffdiff


NCU09196

cuffdiff


NCU11466

cuffdiff


NCU11957


NCU00995
cuffdiff
yes
yes


NCU01720
cuffdiff
yes


NCU03293
cuffdiff
yes
cuffdiff


NCU04169

yes


NCU04170

yes


NCU04467
cuffdiff
yes
cuffdiff


NCU04932
cuffdiff
yes


NCU04998

yes


NCU05134
yes
yes
yes


NCU05350

yes
cuffdiff


NCU05829
cuffdiff
yes


NCU05852

yes
cuffdiff


NCU05908

cuffdiff
yes


NCU06143

yes
cuffdiff


NCU06983

yes


NCU06991

yes


NCU08635
yes
yes


NCU09046

yes


NCU09172

cuffdiff


NCU09424

yes


NCU09498
cuffdiff
yes


NCU09823
cuffdiff
yes
cuffdiff


NCU09848
cuffdiff
yes


NCU10014
cuffdiff
yes
cuffdiff


NCU10039

yes


NCU10687
cuffdiff
yes


NCU00561

yes


NCU00859
cuffdiff
yes
cuffdiff


NCU02042
cuffdiff
yes


NCU02164
cuffdiff
yes


NCU04482

yes


NCU04486

yes


NCU05236

yes


NCU05761

yes


NCU05763

yes


NCU06328
cuffdiff
yes


NCU07948

yes


NCU08140


NCU08447

yes


NCU09734

yes


NCU12011

yes


NCU00408
cuffdiff


NCU00633
cuffdiff

cuffdiff


NCU00870

yes
cuffdiff


NCU00871


NCU00965


NCU01003


NCU01049


NCU01077


NCU01148


NCU01944

yes


NCU01970
cuffdiff
cuffdiff


NCU01983
cuffdiff


NCU02008
yes
cuffdiff


NCU02061
yes
yes
cuffdiff


NCU02600


NCU02625


NCU02720
cuffdiff


NCU02915

cuffdiff


NCU03152
cuffdiff


NCU03329
cuffdiff
cuffdiff


NCU03433


NCU04127
cuffdiff


NCU04522

cuffdiff
yes


NCU04830
cuffdiff


NCU04905

cuffdiff


NCU05056


NCU05170
cuffdiff
cuffdiff
cuffdiff


NCU05569
cuffdiff
cuffdiff


NCU05574

cuffdiff
yes


NCU05846

cuffdiff
yes


NCU05848
cuffdiff
cuffdiff
cuffdiff


NCU05854


NCU06214
cuffdiff
cuffdiff


NCU06312


NCU06704
cuffdiff
cuffdiff
cuffdiff


NCU07207


NCU07336


NCU07339


NCU07453
cuffdiff


NCU07897


NCU07979
yes


NCU08043


NCU08113

cuffdiff
yes


NCU08117


NCU08379
cuffdiff
cuffdiff
cuffdiff


NCU08624


NCU08784


NCU09003


NCU09426


NCU09479


NCU09522


NCU09523


NCU09689

cuffdiff
yes


NCU10521
cuffdiff


NCU11118
cuffdiff

cuffdiff


NCU11278


NCU11327
cuffdiff


NCU11397
cuffdiff


NCU11690


NCU11722
cuffdiff


NCU11862

cuffdiff


NCU00247
yes

cuffdiff


NCU01347


cuffdiff


NCU01598


NCU03761
cuffdiff

yes


NCU04635
yes
cuffdiff
yes


NCU04667
cuffdiff
cuffdiff


NCU05058


NCU05128

yes


NCU06265
yes
cuffdiff
cuffdiff


NCU06615
cuffdiff
cuffdiff
cuffdiff


NCU06895

cuffdiff
cuffdiff


NCU07233
cuffdiff
cuffdiff
cuffdiff


NCU07423


cuffdiff


NCU07424
cuffdiff
cuffdiff
cuffdiff


NCU07895

cuffdiff
cuffdiff


NCU08418

cuffdiff
cuffdiff


NCU08557
yes


NCU08712
cuffdiff
cuffdiff
cuffdiff


NCU09060


yes


NCU09231

cuffdiff


NCU09685


yes


NCU09958
cuffdiff
cuffdiff
yes


NCU10276
cuffdiff
yes
cuffdiff


NCU11697


cuffdiff


NCU11944


NCU12051

cuffdiff


NCU12128


NCU12145

cuffdiff


NCU00289
cuffdiff
cuffdiff
cuffdiff


NCU00496


NCU00763


NCU01386
cuffdiff
cuffdiff
cuffdiff


NCU02485

yes
cuffdiff


NCU02882

yes


NCU04618
yes
cuffdiff


NCU04871


NCU04904
cuffdiff
cuffdiff


NCU05351

cuffdiff


NCU05501

yes


NCU05906

cuffdiff


NCU06373

cuffdiff


NCU07270


NCU08116


NCU08397
cuffdiff
yes
yes


NCU08748
cuffdiff


NCU08867

yes


NCU09176
yes
yes
cuffdiff


NCU11769


NCU11828
cuffdiff
yes


NCU11905


NCU00011


NCU00397
cuffdiff
cuffdiff


NCU00510

cuffdiff


NCU00935
cuffdiff
yes


NCU01880

cuffdiff


NCU02080
cuffdiff
cuffdiff
cuffdiff


NCU02130

cuffdiff


NCU02163
cuffdiff
yes
cuffdiff


NCU02365

yes


NCU03157

cuffdiff


NCU03352
cuffdiff
cuffdiff


NCU03398

cuffdiff
cuffdiff


NCU03570

yes


NCU04282

yes


NCU04342
cuffdiff
cuffdiff
cuffdiff


NCU04360
cuffdiff
cuffdiff


NCU04525
cuffdiff
yes


NCU04866
cuffdiff
yes


NCU05784

yes


NCU05951

yes


NCU05976


NCU06156
cuffdiff
cuffdiff


NCU06986

yes


NCU07126
cuffdiff
cuffdiff


NCU07593


NCU07718
cuffdiff
yes


NCU08224

yes


NCU08469

cuffdiff


NCU08726

cuffdiff


NCU09049

yes


NCU09115
cuffdiff
cuffdiff
cuffdiff


NCU09883

cuffdiff


NCU10658

cuffdiff


NCU10770


NCU11294
cuffdiff
cuffdiff
cuffdiff


NCU00304
cuffdiff
yes
cuffdiff


NCU00798
cuffdiff
yes


NCU01136
cuffdiff
yes


NCU01430

yes


NCU03791
cuffdiff
yes
cuffdiff


NCU04167

yes
cuffdiff


NCU04400
cuffdiff
yes
cuffdiff


NCU04557

yes


NCU04879
cuffdiff
yes
yes


NCU04910
cuffdiff
cuffdiff
cuffdiff


NCU04928
cuffdiff


NCU05068

yes
cuffdiff


NCU05755

yes


NCU05826
cuffdiff
yes


NCU05832
cuffdiff
yes
cuffdiff


NCU05875

yes


NCU05909

yes


NCU06181

yes


NCU06235
cuffdiff
yes


NCU06387

yes


NCU07235

yes


NCU07510

yes


NCU07572

yes
cuffdiff


NCU07997
cuffdiff
yes
cuffdiff


NCU08383

yes


NCU08491

cuffdiff
cuffdiff


NCU08634

yes


NCU09075
yes
yes


NCU09415
cuffdiff
yes


NCU09856

yes


NCU09874
cuffdiff
yes
yes


NCU09906
yes
yes


NCU10284
cuffdiff
yes
cuffdiff


NCU10697

yes


NCU11095

yes


NCU11291
cuffdiff
yes


NCU11689

yes


NCU11801

yes


NCU11932

yes
cuffdiff


NCU00365
cuffdiff
yes


NCU00375

yes


NCU00755

yes


NCU01109

yes


NCU01292
cuffdiff


NCU01551

yes


NCU01649
cuffdiff
cuffdiff


NCU03011

yes


NCU03417

yes


NCU04285

yes


NCU04843
cuffdiff
cuffdiff
cuffdiff


NCU04851

cuffdiff


NCU04861

yes


NCU04862
yes
yes


NCU05006
cuffdiff
yes


NCU05189

yes


NCU05197
cuffdiff
cuffdiff


NCU05477

yes


NCU05762

yes


NCU05764

yes


NCU05766
cuffdiff
yes


NCU05859
cuffdiff
yes


NCU05933

cuffdiff


NCU06334

yes


NCU07180

yes


NCU07363
cuffdiff
yes


NCU08037
cuffdiff
yes
cuffdiff


NCU08155
cuffdiff
yes
cuffdiff


NCU08156

yes


NCU08170

yes


NCU08455

yes


NCU08554

cuffdiff
cuffdiff


NCU08622
cuffdiff
yes


NCU08700

yes


NCU08775

yes


NCU09272

yes


NCU09273
yes
yes


NCU09274
yes
yes


NCU09335

yes
cuffdiff


NCU09342

yes


NCU09714
yes
cuffdiff


NCU09782
cuffdiff
cuffdiff
cuffdiff


NCU10062
cuffdiff
cuffdiff


NCU10301

cuffdiff


NCU11565

cuffdiff


NCU11774
cuffdiff
cuffdiff


NCU11881
cuffdiff
yes


NCU11974

yes
cuffdiff


NCU11989

yes


NCU12012

yes


NCU12014

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

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 is a clr-2 transcription factor protein, selected from the group consisting 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), 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), XP_384856.1 (SEQ ID NO: 81), 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_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), EFQ30604.1 (SEQ ID NO: 68), Podospora_anserina_S_mat+(SEQ ID NO: 225), and Leptosphaeria_maculans_JN3 (SEQ ID NO: 233); 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 fungal host cell is incubated under conditions sufficient for the fungal host cell to express said transcription factor protein.

3. The method of claim 1, 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.

4. The method of claim 1, wherein said cellulose-containing material comprises biomass.

5. The method of claim 4, wherein said biomass 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.

6. The method of claim 1, 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 and further comprising the step of 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.

7. The method of claim 6, wherein said biofuel is selected from the group consisting of ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.

8. The method claim 1, 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.

9. The method claim 1, wherein said at least one recombinant nucleic acid is SEQ ID NO: 5.

10. The method claim 1, 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 is a clr-1 transcription factor protein, selected from the group consisting 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), 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), 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), EG059041.1 (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), Trichoderma reesei clr-1 (SEQ ID NO: 182), and NCU00808 (SEQ ID NO: 213).

11. The method claim 10, wherein the at least one additional recombinant nucleic acid encoding said additional transcription factor protein is SEQ ID NO: 2.

12. The method claim 1, wherein said fungal host cell further comprises at least one recombinant nucleic acid encoding a hemicellulase.

13. The method of claim 1, 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 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), and Chrysosporium lucknowense.

14. 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 is a clr-2 transcription factor protein, selected from the group consisting 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), 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), XP_384856.1 (SEQ ID NO: 81), 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_751092.1 (SEQ ID NO: 66), AN6832 (SEQ ID NO: 67), EFQ30604.1 (SEQ ID NO: 68), Podospora_anserina_S_mat+(SEQ ID NO: 225), and Leptosphaeria_maculans_JN3 (SEQ ID NO: 233); 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.

15. The method of claim 14, wherein said fungal host cell is cultured in the absence of cellulose.