Yeast Expressing Cellulases For Simultaneous Saccharification And Fermentation Using Cellulose

Abstract

The present invention is directed to cellulytic host cells. The host cells of the invention expressing heterologous cellulases and are able to produce ethanol from cellulose. According to the invention, host cells expressing a combination of heterologous cellulases can be used to produce ethanol from cellulose. In addition, multiple host cells expressing different heterologous cellulases can be co-cultured together and used to produce ethanol from cellulose. Furthermore, the invention demonstrates for the first time the ability of Kluyveromyces to produce ethanol from cellulose. The yeast strains and co-cultures of yeast strains of the invention can be used to produce ethanol on their own, or can also be used in combination with externally added cellulases to increase the efficiency of saccharification and fermentation processes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser. No. 13/130,549, filed Feb. 7, 2012, which is a '371 National Stage Application of International Application No. PCT/US2009/065571, filed Nov. 23, 2009, which claims the benefit of U.S. Provisional Application No. 61/116,981, filed Nov. 21, 2008. The entire contents of each application are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 23, 2015, is named 191SeqList.txt and is 169,788 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Lignocellulosic biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the cellulose and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic.

[0004] Biologically mediated processes are promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.

[0005] CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.

[0006] Three major types of enzymatic activities are required for native cellulose degradation: The first type are endoglucanases (1,4-.beta.-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (1,4-.beta.-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-.beta.-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. The third type are .beta.-glucosidases (.beta.-glucoside glucohydrolases; EC 3.2.1.21). .beta.-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units.

[0007] Bakers' yeast (Saccharomyces cerevisiae) remains the preferred micro-organism for the production of ethanol (Hahn-Hagerdal, B., et al., Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Favorable attributes of this microbe include (i) high productivity at close to theoretical yields (0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, and (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making, and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolyzates resulting from biomass pretreatment.

[0008] One major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins. In attempt to address this problem, several heterologous cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the degradation of cellulosic derivatives (Van Rensburg, P., et al., Yeast 14, 67-76 (1998)), or growth on cellobiose (Van Rooyen, R., et al., J. Biotech. 120, 284-295 (2005)); McBride, J. E., et al., Enzyme Microb. Techol. 37, 93-101 (2005)). However, current levels of expression and specific activity of cellulases heterologously expressed in yeast are still not sufficient to enable efficient growth and ethanol production by yeast on cellulosic substrates without externally added enzymes. There remains a significant need for improvement in the amount of cellulase activity in order to attain the goal of achieving a consolidated bioprocessing (CBP) system capable of efficiently and cost-effectively converting cellulosic substrates to ethanol.

[0009] Another major shortcoming of the use of S. cerevisiae is that externally produced cellulases function optimally at a higher temperature than the temperature at which S. cerevisiae function optimally. Thus, either the processing must be carried out in a two step process at two different temperatures or one temperature can be selected where both processes function to some extent, but at least one of the processes does not occur at optimal efficiency.

[0010] In order to address these limitations, the present invention provides for heterologous expression of wild-type and codon-optimized combinations of heterologous cellulases in yeast that allows efficient production of ethanol from cellulose sources. The invention also provides for expression of such heterologous cellulases in thermotolerant yeast and methods of using such transformed yeast for ethanol production.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The present invention is directed to cellulytic host cells. The host cells of the invention expressing heterologous cellulases and are able to produce ethanol from cellulose.

[0012] In particular, in some embodiments, the invention provides a transformed thermotolerant yeast host cell comprising at least one heterologous polynucleotide comprising a nucleic acid encoding a cellulase, wherein the yeast host cell is capable of producing ethanol when grown using cellulose as a carbon source.

[0013] In another embodiment, the invention provides a transformed thermotolerant yeast host cell comprising: (a) at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase; (b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a .beta.-glucosidase; (c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a first cellobiohydrolase; and (d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a second cellobiohydrolase.

[0014] In another embodiment, the invention provides a transformed yeast host cell comprising: (a) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is an endoglucanase; (b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a .beta.-glucosidase; (c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a first cellobiohydrolase; and (d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a second cellobiohydrolase, wherein at least two of the cellulases are secreted by the cell.

[0015] In yet another embodiment, the invention provides a transformed yeast host cell comprising at least six heterologous polynucleotides, wherein each heterologous polynucleotide comprises a nucleic acid which encodes a cellulase.

[0016] In yet another embodiment, the invention provides a transformed yeast host cell comprising at least four heterologous polynucleotides, wherein each heterologous polynucleotide comprises a nucleic acid which encodes an endogluconase.

[0017] In still another embodiment, the invention provides a co-culture comprising at least two yeast host cells wherein (a) at least one of the host cells comprises a first heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is an endoglucanase; (b) at least one of the host cells comprises a second heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a .beta.-glucosidase; (c) at least one of the host cells comprises a third heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a first cellobiohydrolase; (d) at least one of the host cells comprises a fourth heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a second cellobiohydrolase; wherein the first polynucleotide, the second polynucleotide, the third polynucleotide and the fourth polynucleotide are not in the same host cell; and wherein the co-culture is capable of producing ethanol from cellulose.

[0018] In some particular embodiments of the invention, the cellulose carbon source is insoluble cellulose, crystalline cellulose, cellulose derived from lignocellulose, hardwood, phosphoric acid swollen cellulose or microcrystalline cellulose.

[0019] In some embodiments, the host cells of the invention comprise a heterologous polynucleotide comprising a nucleic acid encoding a first cellobiohydrolase, a polynucleotide comprising a nucleic acid encoding an endoglucanase, a polynucleotide comprising a nucleic acid encoding a .beta.-glucosidase and/or a polynucleotide comprising a nucleic acid encoding a second cellobiohydrolase.

[0020] In some embodiments, the cellulase, endoglucanase, .beta.-glucosidase or cellobiohydrolase is a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridium thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinonmyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, or Arabidopsis thaliana cellulase, endoglucanase, .beta.-glucosidase or cellobiohydrolase.

[0021] In some particular embodiments, the cellobiohydrolase is an H. grisea CBH1, a T. aurantiacus CBH1, a T. emersonii CBH1, a T. reesei CBH1, a T. emersonii CBH2, a C. lucknowense CBH2 or a T. reesei CBH2. In some embodiments, the heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase, encodes a fusion protein comprising a cellobiohydrolase and a cellulose binding module (CBM). In some particular embodiments, the CBM is the CBM of T. reesei CBH2, the CBM of T. reesei CBH1 or the CBM of C. lucknowense CBH2b. In some particular embodiments, the CBM is fused to the cellobiohydrolase via a linker sequence. In some particular embodiments, the host cell expresses a first and a second cellobiohydrolase, wherein the first cellobiohydrolase is a T. emersonii CBH1 and CBD fusion, and the second cellobiohydrolase is a C. lucknowense CBI 12b.

[0022] In other particular embodiments, the .beta.-glucosidase is a S. fibuligera .beta.-glucosidase. In another particular embodiment, the endoglucanase is a C. formosanus endoglucanase. In another particular embodiment, the endoglucanase is a T. reesei endoglucanase, e.g. T. reesei EG2.

[0023] In some embodiments of the invention, at least one or at least two of the cellulases is tethered. In other embodiments of the invention, at least one of the cellulases is secreted. In another embodiment, at least one of the cellulases is tethered and at least one of the cellulases is secreted. In another embodiment, all of the cellulases are secreted.

[0024] In some embodiments of the invention, the nucleic acid encoding a cellulase is codon optimized.

[0025] In some embodiments, the host cell can be a thermotolerant host cell. In some embodiments, the host cell is a Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitanine, Candida mexicana, Hansenula polymorpha or Kluyveromyces host cell. For example, in some embodiments, the host cell is a K. lactis or K. marxianus host cell. In some embodiments, the thermotolerant host cell is an S. cerevisiae host cell, wherein the S. cerevisiae is selected to be thermotolerant.

[0026] In some embodiments, the host cell can be an oleaginous yeast cell. In some particular embodiments, the oleaginous yeast cell is a Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon or Yarrowia cell.

[0027] In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

[0028] In some particular embodiments, the host cell can produce ethanol from cellulose at temperatures above about 30.degree. C., 35.degree. C., 37.degree. C., 42.degree. C., 45.degree. C. or 50.degree. C.

[0029] In another particular embodiment, the host cell can produce ethanol at a rate of at least about 10 mg per hour per liter, at least about 30 mg per hour per liter, at least about 40 mg per hour per liter, at least about 50 mg per hour per liter, at least about 60 mg per hour per liter, at least about 70 mg per hour per liter, at least about 80 mg per hour per liter, at least about 90 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, or at least about 1 g per hour per liter.

[0030] The present invention also provides methods of using the host cells and co-cultures of the invention. For example, the present invention is also directed to a method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell or co-culture of the invention. The invention is also directed to a method of fermenting cellulose comprising culturing a host cell or co-culture of the invention in medium that contains insoluble cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose. In some particular embodiments, the methods further comprise contacting the cellulosic substrate with externally produced cellulase enzymes.

[0031] In some particular methods of the invention, the cellulosic substrate is a lignocellulosic biomass selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, Agave, and combinations thereof.

[0032] In some particular methods of the invention, the host cell or co-culture produces ethanol. The ethanol can be produced at a rate of at least about 10 mg per hour per liter, at least about 30 mg per hour per liter, at least about 40 mg per hour per liter, at least about 50 mg per hour per liter, at least about 60 mg per hour per liter, at least about 70 mg per hour per liter, at least about 80 mg per hour per liter, at least about 90 mg per hour per liter, at least about 100 ng per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, or at least about 1 g per hour per liter.

[0033] In other particular methods of the invention, the host cell or co-cultures contact a cellulosic substance at a temperature of at least about 37.degree. C., least about 42.degree. C., from about 42.degree. C. to about 45.degree. C., or from about 42.degree. C. to about 50.degree. C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows an image of a CMC plate assay to detect endoglucanase I activity in K. lactis (colonies numbered 1-8) and K. marxianus strains (colonies numbered 9-16) transformed with heterologous cellulases. Strains 8 and 16 are untransformed negative controls. The plate on the left shows colony growth, and the plate on the right shows CMCase activity, indicated by the presence of a clearance zone. Clearance zones appear as white spots in the image.

[0035] FIG. 2 depicts the results of an MU-lac assay to detect CBH1 activity in K. marxianus strains transformed with heterologous cellulases.

[0036] FIG. 3 depicts the percent of Avicel converted by several strains of K. marxianus expressing heterologous cellulases.

[0037] FIG. 4 depicts the ethanol production/consumption from Avicel by several strains of K. marxianus expressing heterologous cellulases.

[0038] FIG. 5 depicts the growth of S. cerevisiae expressing heterologous cellulases on bacterial microcrystalline cellulose (BMCC).

[0039] FIG. 6 depicts the ethanol production from Avicel by an S. cerevisiae strain expressing heterologous cellulases.

[0040] FIG. 7 depicts the ethanol production from pretreated hardwood (5% based on a dry weight percentage) by an S. cerevisiae strain expressing heterologous cellulases.

[0041] FIG. 8 depicts the ethanol production from pretreated hardwood (5% based on a dry weight percentage) by an S. cerevisiae expressing heterologous cellulases in the presence of various concentrations of exogenously added cellulases.

[0042] FIG. 9 depicts the ethanol production from Avicel by MO288 (circles) and a control strain (triangles) in both YP media and YNB media.

[0043] FIG. 10 depicts the ethanol yield from Avicel (15% based on a dry weight percentage) by a small scale simultaneous saccharification and fermentation (SSF) process using S. cerevisiae supplemented with external cellulases. The yield from a yeast strain expressing heterologous cellulases (MO288) is compared to the yield from a control strain (MO249) at a variety of external cellulase concentrations over 150 hours. (100% cellulase loading indicates 25 mg/g total solids; initial solids concentration was 15%.)

[0044] FIG. 11 depicts the theoretical ethanol yield from a simultaneous saccharification and fermentation (SSF) process using S. cerevisiae supplemented with external cellulases. The yield from a yeast strain expressing heterologous cellulases (MO288) is compared to the yield from a control strain (MO249).

[0045] FIG. 12 illustrates the predicted cellulase enzyme savings based on ethanol yield at 168 hours of simultaneous saccharification and fermentation (SSF) process.

[0046] FIG. 13 shows the activity of an artificial cellulase in the Avicel conversion assay as described in Example 9. The MO429 strain was transformed the CBH1 consensus sequence "CBH1cons." and the MO419 strain was transformed with empty pMU451 vector as a negative control. Descriptions of other strains are found in Table 8 of Example 9.

[0047] FIG. 14 demonstrates the activity of yeast expressing various combinations of CBH1 and CBH2 enzymes on Avicel as described in Example 10.

[0048] FIG. 15 demonstrates the activity of yeast expressing various cellulase enzymes on Avicel as described in Example 10.

[0049] FIG. 16 depicts the ethanol production from Avicel by a co-culture of live S. cerevisiae strains expressing heterologous cellulases.

[0050] FIG. 17 depicts the ethanol production from Avicel by a co-culture of four S. cerevisiae strains expressing heterologous cellulases as well as the ethanol production from strain MO288, which is expressing four cellulases.

[0051] FIG. 18 depicts the ethanol production from Avicel by a co-culture of four S. cerevisiae strains expressing heterologous cellulases in combination with externally added cellulase.

[0052] FIG. 19 depicts the calculated enzyme savings using a co-culture of four S. cerevisiae strains expressing heterologous cellulases or MO288 as compared to untransformed S. cerevisiae.

[0053] FIG. 20 depicts the xylose utilization and ethanol production of M0509 freezer stock, YPX-isolate and YPD-isolate.

[0054] FIG. 21 depicts the growth of M1105 (labeled "colony C2") and MO1046 in the presence of the same medium and 8 g/L acetate at 40.degree. C.

[0055] FIG. 22 depicts the ethanol production by M1105 (triangles) and M1088 (squares) on 18% TS MS419. The experiment with M1105 had 10% lower enzyme dose and half the inoculated cell density, but produced a higher ethanol titer. The experiment with MO1105 was performed at 40.degree. C., and the experiment with M1088 was performed at 35.degree. C.

[0056] FIG. 23 depicts the ethanol production of M1105 where the fermentation was only inoculated with 0.15 g/L DCW and resulted in some sugar accumulation and 29 g/L ethanol.

[0057] FIG. 24 depicts the ethanol production of M1254 is standard IFM (circles) and low ammonium IFM (squares) conditions.

[0058] FIG. 25 depicts the specific growth rate of single colonies compared to M1254 and M1339 on complex xylose medium supplemented with a synthetic inhibitor mixture (which included 8 g/L acetate) at 40.degree. C. The single colonies were screened at the same conditions as the evolution occurred. Colony C1 was renamed M1360.

[0059] FIG. 26 depicts the fermentation performance of M1360 at 40.degree. C. on industrially relevant fermentation medium supplemented with glucose. The fermentation was inoculated with 60 mg/L dry cell weight of M1360.

[0060] FIG. 27 depicts the ethanol production in SSF runs on PHW (18% solids, unwashed MS149) at 35.degree. C. and 40.degree. C. by several strains. All reactions were loaded with 4 mg/g "zoomerase" (Novozyme 22c).

[0061] FIG. 28 depicts cultures spotted on SC.sup.-URA plates containing 0.2% of either CMC or lichenin or barley-.beta.R-glucan. The top two rows of each plate were Y294 based cultures, and the bottom two rows contained MO749 based strains. Numbers indicate the plasmid contained by each strain. pMU471 contains the C.f.EG and served as positive control. Plates were incubated for 24 hours at 30.degree. C. (pictured on the left), after which colonies were washed of and the plates were stained with 0.1% congo red and destained with 1% NaCl (pictured on the right).

[0062] FIG. 29 depicts SDS-PAGE analysis of the supernatants of Cel5 cellulase producing strains. A strain containing a plasmid with no foreign gene was used as reference strain (REF). The strain containing the plasmid pMU471 expressing C.f.EG, the most successful EG previously found was also included.

[0063] FIG. 30 depicts the activity of strains expressing EGs on (A) PASC (2 hours) and (B) avicel (24 hours). A strain containing a plasmid with no foreign gene was used as reference strain (REF) and the strain expressing C.f.EG (pMU471) was included as positive control.

[0064] FIG. 31 depicts the distribution of avicel conversion ability of yeast supernatants from transformation with TrEG2 and additional TeCBH1w/TrCBD. M1088 conversion is presented as a dark vertical line, and the dotted lines flanking this line represent the standard deviation of the measurement.

[0065] FIG. 32 depicts the conversion of Avicel in the HTP avicel assay (48 hour time point) by supernatants of cellulase expressing yeast strains. M0509 is the negative control expressing no cellulases. Strain 1088 is the parental strain expressing only CBH1. CBH2, and BGL, whereas 1179, 1180, and 1181 are transformants of 1088 also expressing TrEG2.

[0066] FIG. 33 depicts ethanol production in paper sludge CBP/SSF with cellulolytic strain M1403 and non-cellulolytic background strain M1254 with various amounts of commercial enzyme supplementation. Experimental conditions: 30% solids fed batch, 10 g/l cell inoculation, pH 5.5 and temperature 40.degree. C., Zoom=Novozymes 22C cellulase preparation, BGL=AB Enzymes EL2008044L BGL preparation, Xyl=AB Enzymes EL2007020L xylanase preparation.

[0067] FIG. 34 depicts fermentation of two types of paper sludge by CBP yeast (M1179) and a control strain M0509, not expressing cellulases. Experimental conditions: 18% solids, cells loaded at 10 or 1 g/L, pH 5.5, Temp: 35 C, 1 mg/g BGL and 1 mg/g Xyl loaded. BGL=AB Enzymes EL2008044L BGL preparation, Xyl=AB Enzymes EL2007020L xylanase preparation.

[0068] FIG. 35 depicts the performance of cellulolytic yeast strain M0963 and non-cellulolytic control strain (M0509) on 22% unwashed solids of pretreated hardwood (PHW) (MS149) at various external cellulase concentrations. Experimental conditions: 22% solids fed batch, pH 5.4, temperature 35.degree. C., all enzyme protein (EP) was "zoomerase" (Novozymes 22C).

[0069] FIG. 36 depicts the performance of cellulolytic yeast strain M1284 on 30% solids of washed pretreated hardwood at various initial cell loadings. Experimental conditions: 30% solids fed batch, pH 5.0, temperature 35.degree. C., 4 mg EP=0.25 mg BGL+0.25 mg Xylanase+0.25 mg Pectinase+3.25 mg Zoomerase, 20 mg EP=1 mg BGL+1 mg Xylanase+1 mg Pectinase+16.7 mg Zoomerase. Zoomerase=Novozymes 22C cellulase preparation, BGL=AB Enzymes EL2008044L BGL preparation, Xyl=AB Enzymes EL2007020L xylanase preparation, Pectinase=Genencor Multifect pectinase FE.

[0070] FIG. 37 depicts the ethanol production in washed corn stover CBP/SSF with cellulolytic strain M1284 and non-cellulolytic background strain M0509 with various amounts of commercial enzyme supplementation. Experimental conditions: 18% solids fed batch, 10 g/l cell inoculation, pH 5.0 and temperature 35.degree. C., 1 mg/g BGL and 1 mg/g xylanase loaded in each case. BGL=AB Enzymes EL2008044L BGL preparation, Xyl=AB Enzymes EL2007020L xylanase preparation.

[0071] FIG. 38A depicts the activity on Avicel of yeast culture supernatants expressing different CBH1 genes. The host strain was either Y294 or M0749. The CBH1 genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0072] FIG. 38B depicts the activity on Avicel of yeast culture supernatants expressing different CBH1 genes. The host strain is M0749. The CBH1 genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Tat, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0073] FIG. 38C depicts the activity on MULac of yeast culture supernatants expressing different CBH1 genes. The host strain is Y294. The CBH1 genes are: Te, Talaromyces emersonii; Ct. Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0074] FIG. 38D depicts the activity on MULac of yeast culture supernatants expressing different CBH1 genes. The host strain is M0749. The CBH1 genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0075] FIG. 38E depicts the activity on estimated CBH1 concentration (mg/L) based on MULac. The host strain is Y294. The CBH1 genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0076] FIG. 38F depicts the activity on estimated CBH1 concentration (mg/L) based on MULac. The host is M0749. The CBH1 genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus. The plasmid names are indicated. Yeast were cultivated in YPD in triplicate for 3 days. The data are means.+-.standard deviation.

[0077] FIG. 39 shows the genes modified in yeast strain M0509.

[0078] FIG. 40 shows the yeast strains used to construct M0509 and the relevant genetic modifications.

[0079] FIG. 41 shows the genealogy of yeast strain M1105.

[0080] FIG. 42 shows the genealogy of yeast strain M1254.

DETAILED DESCRIPTION OF THE INVENTION

[0081] The disclosed methods and materials are useful generally in the field of engineered yeast.

Definitions

[0082] A "vector," e.g., a "plasmid" or "YAC" (yeast artificial chromosome) refers to an extrachromosomal element often carrying one or more genes that are not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. Preferably, the plasmids or vectors of the present invention are stable and self-replicating.

[0083] An "expression vector" is a vector that is capable of directing the expression of genes to which it is operably associated.

[0084] The term "heterologous" as used herein refers to an element of a vector, plasmid or host cell that is derived from a source other than the endogenous source. Thus, for example, a heterologous sequence could be a sequence that is derived from a different gene or plasmid from the same host, from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications). The term "heterologous" is also used synonymously herein with the term "exogenous."

[0085] The term "domain" as used herein refers to a part of a molecule or structure that shares common physical or chemical features, for example hydrophobic, polar, globular, helical domains or properties, e.g., a DNA binding domain or an ATP binding domain. Domains can be identified by their homology to conserved structural or functional motifs. Examples of cellobiohydrolase (CBH) domains include the catalytic domain (CD) and the cellulose binding domain (CBD).

[0086] A "nucleic acid," "polynucleotide," or "nucleic acid molecule" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.

[0087] An "isolated nucleic acid molecule" or "isolated nucleic acid fragment" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0088] A "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific protein, including intervening sequences (introns) between individual coding segments (exons), as well as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences.

[0089] A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified, e.g., in Sambrook, J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (hereinafter "Maniatis", entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of conditions uses a series of washes starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then repeated twice with 0.2.times.SSC, (0.5% SDS at 50.degree. C. for 30 min. For more stringent conditions, washes are performed at higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2.times.SSC, 0.5% SDS are increased to 60.degree. C. Another set of highly stringent conditions uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C. An additional set of highly stringent conditions are defined by hybridization at 0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS.

[0090] Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see. e.g., Maniatis at 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see, e.g., Maniatis, at 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

[0091] The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.

[0092] As known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.

[0093] "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS, 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0094] Suitable nucleic acid sequences or fragments thereof (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% to 75% identical to the amino acid sequences reported herein, at least about 80%, 85%, or 90% identical to the amino acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments are at least about 70%, 75%, or 80% identical to the nucleic acid sequences reported herein, at least about 80%, 85%, or 90% identical to the nucleic acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities/similarities but typically encode a polypeptide having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids.

[0095] A DNA or RNA "coding region" is a DNA or RNA molecule which is transcribed and/or translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. "Suitable regulatory regions" refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region.

[0096] An "isoform" is a protein that has the same function as another protein but which is encoded by a different gene and may have small differences in its sequence.

[0097] A "paralogue" is a protein encoded by a gene related by duplication within a genome.

[0098] An "orthologue" is gene from a different species that has evolved from a common ancestral gene by speciation. Normally, orthologues retain the same function in the course of evolution as the ancestral gene.

[0099] "Open reading frame" is abbreviated ORF and means a length of nucleic acid, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0100] "Promoter" refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. In general, a coding region is located 3' to a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0101] A coding region is "under the control" of transcriptional and translational control elements in a cell when RNA polymerase transcribes the coding region into mRNA, which is then trans-RNA spliced (if the coding region contains introns) and translated into the protein encoded by the coding region.

[0102] "Transcriptional and translational control regions" are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell. In eukaryotic cells, polyadenylation signals are control regions.

[0103] The term "operably associated" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably associated with a coding region when it is capable of affecting the expression of that coding region (i.e., that the coding region is under the transcriptional control of the promoter). Coding regions can be operably associated to regulatory regions in sense or antisense orientation.

[0104] The term "expression," as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

Host Cells Expressing Heterologous Cellulases

[0105] In order to address the limitations of the previous systems, the present invention provides host cells expressing heterologous cellulases that can be effectively and efficiently utilized to produce ethanol from cellulose. In some embodiments, the host cells can be a yeast. According to the present invention the yeast host cell can be, for example, from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia. Yeast species as host cells may include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, or K. fragilis. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In one particular embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the yeast is a thermotolerant Saccharomyces cerevisiae. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0106] In some embodiments of the present invention, the host cell is an oleaginous cell. According to the present invention, the oleaginous host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. According to the present invention, the oleaginous host cell can be an oleaginous microalgae host cell. For example, the oleaginous microalgae host cell can be from the genera Thraustochytrium or Schizochytrium. Biodiesel could then be produced from the triglyceride produced by the oleaginous organisms using conventional lipid transesterification processes. In some particular embodiments, the oleaginous host cells can be induced to secrete synthesized lipids. Embodiments using oleaginous host cells are advantageous because they can produce biodiesel from lignocellulosic feedstocks which, relative to oilseed substrates, are cheaper, can be grown more densely, show lower life cycle carbon dioxide emissions, and can be cultivated on marginal lands.

[0107] In some embodiments of the present invention, the host cell is a thermotolerant host cell. Thermotolerant host cells can be particularly useful in simultaneous saccharification and fermentation processes by allowing externally produced cellulases and ethanol-producing host cells to perform optimally in similar temperature ranges.

[0108] Thermotolerant host cells of the invention can include, for example, Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenula polymorpha and Kluyveromyces host cells. In some embodiments, the thermotolerant cell is an S. cerevisiae strain, or other yeast strain, that has been adapted to grow in high temperatures, for example, by selection for growth at high temperatures in a cytostat.

[0109] In some particular embodiments of the present invention, the host cell is a Kluyveromyces host cell. For example, the Kluyveromyces host cell can be a K. lactis, K. marxianus, K. blattae, K. phaffii, K. yarrowii, K. aestuarii, K. dobzhanskii, K. wickerhamii K. thermotolerans, or K. waltii host cell. In one embodiment, the host cell is a K. lactis, or K. marxianus host cell. In another embodiment, the host cell is a K. marxianus host cell.

[0110] In some embodiments of the present invention the thermotolerant host cell can grow at temperatures above about 30.degree. C., about 31.degree. C., about 32.degree. C., about 33.degree. C., about 34.degree. C., about 35.degree. C., about 36.degree. C., about 37.degree. C., about 38.degree. C., about 39.degree. C., about 40.degree. C., about 41.degree. C. or about 42.degree. C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30.degree. C., about 31.degree. C., about 32.degree. C., about 33.degree. C. about 34.degree. C., about 35.degree. C. about 36.degree. C., about 37.degree. C., about 38.degree. C., about 39.degree. C., about 40.degree. C., about 41.degree. C., about 42.degree. C., or about 43.degree. C., or about 44.degree. C., or about 45.degree. C., or about 50.degree. C.

[0111] In some embodiments of the present invention, the thermotolerant host cell can grow at temperatures from about 30.degree. C. to 60.degree. C., about 30.degree. C. to 55.degree. C. about 30.degree. C. to 50.degree. C., about 40.degree. C. to 60.degree. C., about 40.degree. C. to 55.degree. C. or about 40.degree. C. to 50.degree. C. In some embodiments of the present invention, the thermotolerant host cell can produce ethanol from cellulose at temperatures from about 30.degree. C. to 60.degree. C., about 30.degree. C. to 55.degree. C., about 30.degree. C. to 50.degree. C., about 40.degree. C. to 60.degree. C., about 40.degree. C. to 55.degree. C. or about 40.degree. C. to 50.degree. C.

[0112] In some methods described herein, the host cell has the ability to metabolize xylose. Detailed information regarding the development of the xylose-utilizing technology can be found in the following publications: Kuyper M et al. FEMS Yeast Res. 4: 655-64 (2004), Kuyper M et al. FEMS Yeast Res. 5:399-409 (2005), and Kuyper M et al. FEMS Yeast Res. 5:925-34 (2005), which are herein incorporated by reference in their entirety. For example, xylose-utilization can be accomplished in S. cerevisiae by heterologously expressing the xylose isomerase gene, XylA, e.g. from the anaerobic fungus Piromyces sp. E2, overexpressing five S. cerevisiae enzymes involved in the conversion of xylulose to glycolytic intermediates (xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) and deleting the GRE3 gene encoding aldose reductase to minimise xylitol production.

[0113] According to the methods described herein, the host cells can contain antibiotic markers or can contain no antibiotic markers.

[0114] Host cells are genetically engineered (transduced or transformed or transfected) with the polynucleotides encoding cellulases of this invention which are described in more detail below. The polynucleotides encoding cellulases can be introduced to the host cell on a vector of the invention, which may be, for example, a cloning vector or an expression vector comprising a sequence encoding a heterologous cellulase. The host cells can comprise polynucleotides of the invention as integrated copies or plasmid copies.

[0115] In certain aspects, the present invention relates to host cells containing the polynucleotide constructs described below. The host cells of the present invention can express one or more heterologous cellulase polypeptides. In some embodiments, the host cell comprises a combination of polynucleotides that encode heterologous cellulases or fragments, variants or derivatives thereof. The host cell can, for example, comprise multiple copies of the same nucleic acid sequence, for example, to increase expression levels, or the host cell can comprise a combination of unique polynucleotides. In other embodiments, the host cell comprises a single polynucleotide that encodes a heterologous cellulase or a fragment, variant or derivative thereof. In particular, such host cells expressing a single heterologous cellulase can be used in co-culture with other host cells of the invention comprising a polynucleotide that encodes at least one other heterologous cellulase or fragment, variant or derivative thereof.

[0116] Introduction of a polynucleotide encoding a heterologous cellulase into a host cell can be done by methods known in the art. Introduction of polynucleotides encoding heterologous cellulases into, for example yeast host cells, can be effected by lithium acetate transformation, spheroplast transformation, or transformation by electroporation, as described in Current Protocols in Molecular Biology, 13.7.1-13.7.10. Introduction of the construct in other host cells can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., et al., Basic Methods in Molecular Biology, (1986)).

[0117] The transformed host cells or cell cultures, as described above, can be examined for endoglucanase, cellobiohydrolase and/or .beta. glucosidase protein content. For the use of secreted heterologous cellulases, protein content can be determined by analyzing the host (e.g., yeast) cell supernatants. In certain embodiments, high molecular weight material can be recovered from the yeast cell supernatant either by acetone precipitation or by buffering the samples with disposable de-salting cartridges. Proteins, including tethered heterologous cellulases, can also be recovered and purified from recombinant yeast cell cultures by methods including spheroplast preparation and lysis, cell disruption using glass beads, and cell disruption using liquid nitrogen for example. Additional protein purification methods include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, gel filtration, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0118] Protein analysis methods include methods such as the traditional Lowry method or the protein assay method according to BioRad's manufacturer's protocol. Using such methods, the protein content of saccharolytic enzymes can be estimated. Additionally, to accurately measure protein concentration a heterologous cellulase can be expressed with a tag, for example a His-tag or HA-tag and purified by standard methods using, for example, antibodies against the tag, a standard nickel resin purification technique or similar approach.

[0119] The transformed host cells or cell cultures, as described above, can be further analyzed for hydrolysis of cellulose (e.g., by a sugar detection assay), for a particular type of cellulase activity (e.g., by measuring the individual endoglucanase, cellobiohydrolase or .beta. glucosidase activity) or for total cellulase activity. Endoglucanase activity can be determined, for example, by measuring an increase of reducing ends in an endoglucanase specific CMC substrate. Cellobiohydrolase activity can be measured, for example, by using insoluble cellulosic substrates such as the amorphous substrate phosphoric acid swollen cellulose (PASC) or microcrystalline cellulose (Avicel) and determining the extent of the substrate's hydrolysis. .beta.-glucosidase activity can be measured by a variety of assays, e.g., using cellobiose.

[0120] A total cellulase activity, which includes the activity of endoglucanase, cellobiohydrolase and .beta.-glucosidase, can hydrolyze crystalline cellulose synergistically. Total cellulase activity can thus be measured using insoluble substrates including pure cellulosic substrates such as Whatman No. 1 filter paper, cotton linter, microcrystalline cellulose, bacterial cellulose, algal cellulose, and cellulose-containing substrates such as dyed cellulose, alpha-cellulose or pretreated lignocellulose. Specific activity of cellulases can also be detected by methods known to one of ordinary skill in the art, such as by the Avicel assay (described supra) that would be normalized by protein (cellulase) concentration measured for the sample.

[0121] One aspect of the invention is thus related to the efficient production of cellulases to aid in the digestion of cellulose and generation of ethanol. A cellulase can be any enzyme involved in cellulase digestion, metabolism and/or hydrolysis, including an endoglucanase, exogluconase, or .beta.-glucosidase.

[0122] In additional embodiments, the transformed host cells or cell cultures are assayed for ethanol production. Ethanol production can be measured by techniques known to one or ordinary skill in the art e.g. by a standard HPLC refractive index method.

Heterologous Cellulases

[0123] According to the present invention the expression of heterologous cellulases in a host cell can be used advantageously to produce ethanol from cellulosic sources. Cellulases from a variety of sources can be heterologously expressed to successfully increase efficiency of ethanol production. For example, the cellulases can be from fungi, bacteria, plant, protozoan or termite sources. In some embodiments, the cellulase is a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermnophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, or Arabidopsis thaliana cellulase.

[0124] In some embodiments of the invention, multiple cellulases from a single organism are co-expressed in the same host cell. In some embodiments of the invention, multiple cellulases from different organisms are co-expressed in the same host cell. In particular, cellulases from two, three, four, five, six, seven, eight, nine or more organisms can be co-expressed in the same host cell. Similarly, the invention can encompass co-cultures of yeast strains, wherein the yeast strains express different cellulases. Co-cultures can include yeast strains expressing heterologous cellulases from the same organisms or from different organisms. Co-cultures can include yeast strains expressing cellulases from two, three, four, five, six, seven, eight, nine or more organisms.

[0125] Cellulases of the present invention include both endoglucanases or exoglucanases. The cellulases can be, for example, endoglucanases, .beta.-glucosidases or cellobiohydrolases.

[0126] In certain embodiments of the invention, the endoglucanase(s) can be an endoglucanase I or an endoglucanase II isoform, paralogue or orthologue. In some embodiments, the endoglucanase expressed by the host cells of the present invention can be recombinant endo-1,4-.beta.-glucanase. In particular embodiments, the endoglucanase is a T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, R. speratus Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomycess, Irpex lacteus, C. lucknowense, C. globosum, Aspergillus terreus, Aspergillus fumigatus, Neurospora crassa or Acremonium thermophilum endoglucanase. In one particular embodiment, the endoglucanase comprises an amino acid sequence selected from SEQ ID NOs: 30-39 or 52-56, as shown in Table 1 below. In certain other embodiments, the endoglucanase comprises an amino acid sequence that is at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 30-39 or 52-56.

[0127] As a practical matter, whether any polypeptide is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of the present invention can be determined conventionally using known computer programs. Methods for determining percent identity, as discussed in more detail below in relation to polynucleotide identity, are also relevant for evaluating polypeptide sequence identity.

[0128] In one particular embodiment, the endoglucanase is an endoglucanase I ("eg1") from Trichoderma reesei. In certain embodiments, the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:39.

[0129] In another particular embodiment, the endoglucanase is an endoglucanase from C. formosanus. In certain embodiments, the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:31.

[0130] In another particular embodiment, the the endoglucanase is an endoglucanase from H. jecorina. In certain embodiments, the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:54.

[0131] In certain embodiments, the .beta.-glucosidase is a .beta.-glucosidase I or a .beta.-glucosidase II isoform, paralogue or orthologue. In certain embodiments of the present invention the .beta.-glucosidase is derived from Saccharomycopsis fibuligera. In particular embodiments, the .beta.-glucosidase comprises an amino acid sequence at least about 70, about 80, about 90), about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:40.

[0132] In certain embodiments of the invention, the cellobiohydrolase(s) can be a cellobiohydrolase I and/or a cellobiohydrolase II isoform, paralogue or orthologue. In one particular embodiment, the cellobiohydrolase comprises an amino acid sequence selected from SEQ ID NOs: 21-29 or 46, as shown in Table 1 below. In particular embodiments of the present invention the cellobiohydrolase is a cellobiohydrolase I or II from Trichoderma reesei. In another embodiment, the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:27 or SEQ ID NO:28.

[0133] In other particular embodiments of the present invention the cellobiohydrolase is a cellobiohydrolase I or II from T. emersonii. In another embodiment, the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:23 or SEQ ID NO:24.

[0134] In another embodiment, the cellobiohydrolase of the invention is a C. lucknowense cellobiohydrolase. In a particular embodiment, the cellobiohydrolase is C. lucknowense cellobiohydrolase Cbh2b. In one embodiment, the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:25.

[0135] In some particular embodiments of the invention, the cellulase comprises a sequence selected from the sequences in Table 1 below. The cellulases of the invention also include cellulases that comprise a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99 or 100% identical to the sequences of Table 1.

[0136] Some embodiments of the invention encompass a polypeptide comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 or more consecutive amino acids of any of SEQ ID NOs:21-40, 46 or 52-56, or domains, fragments, variants, or derivatives thereof.

TABLE-US-00001 TABLE 1 Cellulases used in Examples 1-11 as described below. Donor organism/ Accession number and Gene Codon-Optimized DNA sequence used amino acid sequence Cellobiohydrolases Humicola GAATTCATGAGAACCGCTAAGTTCGCTACCTTGGCTGCCTTGGTTGCCTCT CAA35159 grisea cbh1 GCTGCTGCTCAACAAGCCTGTTCCTTGACTACTGAACGTCACCCATCTTTG MRTAKFATLAALVASAAAQQA TCTTGGAACAAGTGTACTGCTGGTGGTCAATGTCAAACTGTCCAAGCCTCC CSLTTERHPSLSWNKCTAGGQC ATCACTTTGGACTCTAATTGGAGATGGACCCACCAAGTCTCTGGTAGTACT QTVQASITLDSNWRWTHQVSGS AACTGTTACACCGGTAATAAGTGGGACACTTCTATTTGTACTGACGCTAA TNCYTGNKWDTSICTDAKSCAQ GTCTTGTGCTCAAAATTGTTGTGTTGATGGTGCTGATTACACCTCCACTTA NCCVDGADYTSTYGITTNGDSLS TGGTATTACCACCAACGGTGACTCTTTGTCCTTGAAGTTCGTTACTAAAGG LKFVTKGQHSTNVGSRTYLMDG TCAACATTCCACCAACGTCGGTTCTAGAACCTACTTAATGGACGGTGAAG EDKYQTFELLGNEFTFDVDVSNI ACAAGTACCAAACCTTCGAATTGTTGGGTAATGAATTTACCTTCGATGTCG GCGLNGALYFVSMDADGGLSR ATGTGTCTAACATCGGTTGTGGTTTGAACGGTGCTTTATACTTCGTTTCTA YPGNKAGAKYGTGYCDAQCPR TGGACGCCGACGGTGGTTTGTCTCGTTACCCAGGTAATAAGGCTGGTGCCA DIKFINGEANIEGWTGSTNDPNA AGTATGGTACCGGTTACTGTGATGCTCAATGCCCAAGAGACATTAAGTTC GAGRYGTCCSEMDIWEANNMA ATCAACGGTGAAGCTAACATTGAAGGTTGGACTGGTTCTACCAACGACCC TAFTPHPCTIIGQSRCEGDSCGGT AAACGCTGGCGCCGGTAGATACGGTACCTGTTGTTCCGAAATGGACATTT YSNERYAGVCDPDGCDFNSYRQ GGGAAGCCAACAACATGGCTACTGCTTTTACTCCACACCCATGTACCATC GNKTFYGKGMTVDTTKKITVVT ATTGGTCAATCCAGATGTGAAGGTGACTCCTGTGGCGGTACCTACTCCAA QFLKDANGDLGEIKRFYVQDGK CGAAAGATACGCTGGTGTTTGTGATCCAGACGGTTGTGACTTCAACTCCTA IIPNSESTIPGVGGNSITQDWCDR CAGACAAGGTAACAAGACTTTCTATGGTAAGGGTATGACTGTCGATACCA QKVAFGDIDDFNRKGGMKQMG CCAAGAAGATCACCGTCGTCACCCAATTCTTGAAGGACGCTAACGGTGAT KALAGPMVLVMSIWDDHASNM TTAGGTGAAATTAAAAGATTCTACGTCCAAGATGGTAAGATCATCCCAAA LWLDSTFPVDAAGKPGAERGAC CTCGAATCTACCATTCCAGGTGTTGAAGGTAATTCCATCACTCAAGACTG PTTSGVPAEVEAPNSNVVFSN GTGTGACAGACAAAAGGTTGCCTTCGGTGATATTGACGACTTCAACAGAA IRFGPIGSTVAGLPGAGNGGNNG AGGGTGGTATGAAGCAAATGGGTAAGGCTTTGGCCGGTCCAATGGTCTTG GNPPPPTTTTSSAPATTTTASAGP GTTATGTCTATTTGGGACGATCACGCTTCCAACATGTTGTGGTTGGACTCC KAGRWQQCGGIGFTGPTQCEEP ACCTTCCCAGTTGATGCTGCTGGTAAGCCAGGTGCCGAAAGAGGTGCTTG YICTKLNDWYSQCL TCCAACTACTTCCGGTGTCCCAGCTGAAGTTGAAGCCGAAGCTCCAAATT (SEQ ID NO: 21) CTAACGTTGTCTTCTCTAACATCAGATTCGGTCCAATCGGTTCCACAGTCG CTGGTTTGCCAGGTGCTGGTAATGGTGGTAATAACGGTGGTAACCCACCA CCACCAACCACTACCACTTCTTCTGCCCCAGCTACTACCACCACCGCTTCT GCTGGTCCAAAGGCTGGTAGATGGCAACAATGTGGTGGTATTGGTTTCAC CGGTCCAACCCAATGTGAAGAACCATACATCTGTACCAAGTTGAACGACT GGTACTCTCAATGTTTATAACTCGAG (SEQ ID NO: 1) Thermoascus GAATTCATGTACCAAAGAGCTCTATTGTTCTCCTTCTTCTTGGCCGCCGCT AAL83303 aurantiacus AGAGCTCATGAAGCCGGTACTGTCACCGCCGAAAACCACCCATCCTTGAC MYRQRALLFSFFLAAARAHEAGT cbh1 TTGGCAACAATGTTCCTCTGGTGGTTCTTGTACTACTCAAAACGGGAAGGT VTAENHPSLTWQQCSSGGSCTT TGTTATTGACGCTAACTGGAGATGGGTTCACACTACCTCCGGTTACACCAA QNGKVVIDANWRWVHTTSGYT CTGTTACACTGGTAACACTTGGGATACTTCCATCTGTCCAGACGACGTTAC NCYTGNTWDTSICPDDVTCAQN CTGTGCTCAAAACTGTGCTTTGGACGGTGCTGACTACTCCGGTACTTACGG CALDGADYSGTYGVTTSGNALR TGTCACTACCTCTGGCAACGCGTTGAGATTGAACTTCGTCACCCAATCTTC LNFVTQSSGKNIGSRLYLLQDDT TGGTAAGAACATCGGTTCTAGATTGTACTTGTTGCAAGACGATACTACTTA TYQIFKLLGQEFTFDVDVSNLPC CCAAATCTTCAAGTTGTTGGGTCAAGAGTTCACTTTCGACGTTGATGTTTC GLNGALYFVAMDADGNLSKYP CAACTTGCCTTGTGGTTTGAACGGTGCTTTGTACTTCGTTGCTATGGACGC GNKAGAKYGTGYCDSQCPRDL CGACGGTAACTTATCCAAGTACCCAGGTAACAAGGCCGGTGCCAAGTACG KFINGQNVEGWQPSANDPNAG GTACCGGTTACTGTGATTCTCAATGTCCAAGAGACCTAAAATTCATTAACG VGNHGSSCAEMDVWEANSISTA GTCAAGCTAACGTCGAAGGTTGGCAACCATCTGCTAACGATCCAAACGCC VTPHPCDTPGQTMCQGDDCGGT GGTGTCGGTAATCACGGTTCCTCCTGTGCTGAAATGGACGTTTGGGAAGC YSSTRYAGTCDTDGCDFNPYQP TAACTCTATCTCCACCGCCGTCACTCCACATCCATGTGATACCCCAGGTCA GNHSFYGPGKIVDTSSKFTVVTQ AACCATGTGTCAAGGTGATGATTGTGGTGGTACCTACTCTTCCACTAGATA FITDDGTPSGTLTEIKRFYVQNG CGCTGGTACCTGTGACACCGACGGTTGTGATTTCAACCCATACCAACCAG KVIPQSESTISGVTGNSITTEYCT GTAACCACTCTTTCTACGGTCCAGGTAAGATTGTCGATACTTCTTCTAAGT AQKAAFDNTGFFTIIGGLQKISQ TCACTGTTGTCACTCAATTCATTACCGACGATGGTACCCCATCTGGTACCC ALAQGMVLVMSLWDDHAANM TAACTGAAATTAAGAGATTCTACGTCCAAAACGGTAAAGTCATTCCACAA LWLDSTYPTDADPDTPGVARGT TCCGAAAGCACCATTTCCGGTGTTACCGGTAACTCCATCACCACTGAATAC CPTTSGVPADVESQNPNSYVIYS TGTACCGCTCAAAAGGCCGCCTTTGACAACACCGGTTTCTTCACCCATGGT NIKVGPINSTFTAN GGTTTGCAAAAGATTTCTCAAGCCTTGGCTCAAGGTATGGTTTTGGTCATG (SEQ ID NO: 22) TCCTTGTGGGATGACCACGCTGCTAACATGTTGTGGTTGGATTCTACTTAC CCAACTGACGCTGATCCAGACACCCCAGGTGTTGCTAGAGGTACTTGTCC AACCACTTCTGGTGTTCCAGCTGACGTCGAATCTCAAAACCCTAACTCTTA CGTTATCTACTCTAACATCAAGGTGGGTCCAATTAACTCCACCTTCACTGC TAACTAACTCGAG (SEQ ID NO: 2) Talaromyces GAATTCATGCTAAGAAGAGCTTTACTATTGAGCTCTTCTGCTATCTTGGCC AAL89553 emersonii GTTAAGGCTCAACAAGCCGGTACCGCTACTGCTGAAAACCACCCTCCATT MLRRALLISSSAILAVKAQQAG cbh1 GACCTGGCAAGAATGTACCGCTCCAGGTTCTTGTACCACCCAAAACGGTG TATAENHPPLTWQECTAPGSCTT GACCTGGCAAGAATGTACCGCTCCAGGTTCTTGTACCACCCAAAACGGTG QNGAVVLDANWRWVHDVNGY CTGTCGTCTTGGACGCTAACTGGAGATGGGTCCACGACGTCAACGGTTAC TNCYTGNTWDPTYCPDDETCAQ ACTAACTGTTACACCGGTAACACCTGGGACCCAACTTACTGTCCAGACGA NCALDGADYEGTYGVTSSGSSL CGAAACTTGCGCTCAAAACTGTGCCTTGGACGGTGCTGACTACGAAGGTAC KLNFVTGSNVGSRLYLLQDDST TTACGGTGTTACCTCCTCTGGTTCTTCCTTGAAGTTGAACTTCGTCACTGG YQIFKLLNREFSFDVDVSNLPCG TTCTAACGTCGGTTCCAGATTGTATTTGTTGCAAGATGACTCCACTTACCA LNGALYFVAMDADGGVSKYPN AATCTTCAAGTTGTTGAACAGAGAATTTTCTTTCGACGTCGATGTGTCCAA NKAGAKYGTGYCDSQCPRDLKF CTTGCCTTGTGGTTTGAACGGTGCTCTATACTTCGTTGCTATGGACGCTGA IDGEANVEGWQPSSNNANTGIG TGGTGGTGTTTCCAAGTACCCAAACAACAAGGCTGGTGCCAAATACGGTA DHGSCCAEMDVWEANSISNAVT CTGGTTACTGTGACTCTCAATGTCCACGTGACTTGAAGTTTATTGATGGTG PHPCDTPGQTMCSGDDCGGTYS AAGCTAATGTCGAAGGTTGGCAACCATCTTCTAACAACGCTAACACTGGC NDRYAGTCDPDGCDFNPYRMG ATCGGTGACCACGGTTCTTGCTGTGCCGAAATGGACGTTTGGGAAGCCAA NTSFYGPGKIIDTTKP CTCCATTTCCAACGCCGTCACTCCACACCCATGTGACACTCCAGGTCAAAC FTVVTQFLTDDGTDTGLSEIKR TATGTGTTCCGGCGATGACTGTGGTGGTACTTACTCTAACGATAGATACGC FYIQNSNVIPQPNSDISGVTGNSI TGGTACCTGTGATCCAGACGGTTGCGACTTCAATCCATACAGAATGGGTA TTEFCTAQKQAFGDTDDFSQHG ACACTTCCTTTTACGGTCCAGGCAAGATCATCGACACTACTAAGCCATTCA GLAKMGAAMQQGMVLVMSLW CTGTTGTCACCCAATTCTTGACCGACGATGGTACTGATACCGGTACTTTGT DDYAAQMLWLDSDYPTDADPT CCGAAATCAAGAGATTCTACATCCAAAACTCTAACGTCATCCCACAACCA TPGIARGTCPTDSGVPSDVESQSP AATTCCGACATCTCTGGTGTCACTGGTAACTCCATTACCACCGAATTTTGT NSYVTYSNIKFGPINSTFTAS ACCGCCCAAAAGCAAGCTTTCGGTGACACCGACGACTTCTCTCAACACGG (SEQ ID NO: 23) TGGTTTGGCTAAGATGGGTGCTGCTATGCAACAAGGTATGGTTTTGGTCAT GTCTTTGTGGGACGACTACGCTGCTCAAATGTTGTGGTTGGACTCCGATTA CCCAACCGATGCCGACCCAACCACCCCTGGTATCGCTAGAGGTACCTGTC CAACTGACTCTGGTGTTCCATCTGACGTCGAATCCCAATCTCCAAACTCCT ACGTCACTTACTCCAACATTAAATTCGGTCCAATCAACTCCACTTTCACTG CTTCTTAACTCGAG (SEQ ID NO: 3) Talaromyces GAATTCATGCGTAACTTGTTGGCCTTGGCTCCAGCCGCTTTGTTGGTTGGT AAL78165 emersonii GCTGCCGAAGCTCAACAATCCTTGTGGGGTCAATGCGGTGGTTCCTCCTG MRNLLALAPAALLVGAAEAQQS cbh2 GACTGGTGCAACTTCCTGTGCCGCTGGTGCCACCTGTTCCACCATTAACCC LWGQCGSSWTGATSCAAGAT ATACTACGCTCAATGTGTTCCAGCCACTGCCACTCCAACTACCTTGACTAC CSTINPYYAQCVPATATPTTLTT CACCACTAAGCCAACCTCCACGGTGGTGCTGCTCCAACCACTCCACCACC TTKPTSTGGAAPTTPPPTTTGTTT AACTACTACCGGTACTACCACCTCTCCAGTCGTCACCAAGACCTGCCTCCG SPVVTRPASASGNPFEGYQLYAN CCTCCGGTAATCCATTCGAAGGTTATCAATTGTACGCTAACCCTTACTACG PYYASEVISLAIPSLSSELVPKAS CTTCTGAAGTCATTTGGCTATCCCATCTTTGAGCTCCGAGTTGGTCCC EVAKVPSFVWLDQAAKVPSMG AAAGGCCTCCGAAGTTGCTAAGGTCCCTTCATTTGTCTGGTTAGATCAAGC DYLKDIQSQNAAGADPPIAGIFV TGCCAAGGTTCCATCTATGGGTGATTACTTGAAGGATATTCAATCTCAAAA VYDLPDRDCAAAASNGEFSIAN CGCTGCTGGTGCTGATCCACCAATCGCCGGTATTTTCGTTGTTTACGATT NGVALYKQYIDSIREQLTTYSDV GCCAGATAGAGACTGTGCCGCCGCTGCTTCAACGGTGAATTTTCTATCGC IITLVIEPDSLANVVTNLNVPKC CAACAACGGTGTCGTTTATACAAACAATATATCGATTCCATTAGAGAAC ANAQDAYLECINYAITQLDLPNV AATTAACCACTTACTCCGACGTCCATACCATCTTGGTTATCGAACCAGACT AMYLDAGHAGWLWQANLAP CTTTGGCTAACGTTGTCACTAACTTGAACGTTCCAAAATGTGCTAACGTC AAQLFASVYKNASSPASVRGLA AAGATGCTTACTTGGAATGTATCAACTACGCTATTACCCAATTGGACTTGC TNVANYNAWSISRCPSYTQGDA CAAACGTTGCTATGTACTTGGACGTGGTCACGCCGGTTGGTTGGGTTGGCA NCDEEDYVNALGPLFQEQGFPA AGCCAACTTGGCCCCCAGCTGCTCAATTATTCGCTTCTGTTTACAAGAACG YFIIDTSRNGVRPTKQSQWGDW CCTCTTCCCCAGCCTCTGTTAGAGGTTTGGCTACCAACGTGGCTAACTACA CNVIGTGFGVRPTTDTGNPLEDA ACGCCTGGTCCATTTCTAGATGTCCATCCTACACTCAAGGTGACGATAACT FVWVKPGGESDGTSNTTSPRYD GTGATGAAGAAGATTACGTTAACGCTTTGGGTCCATTGTTCCAAGAACAA YIICGLSDALQPAPEAGTWFQAY GGTTTCCCAGCTTACTTCATCATCGACACTTCCCGTAACGGTGTCAGACCA FEQLLTNANPLF ACTAAGCAATCTCAATGGGGTGACTGGTGTAACGTTATTGGTACCCGTTC (SEQ ID NO: 24) GGTGTTAGACCAACCACCGACACTGGTAACCCATTGGAAGACGCTTTGT TTGGGTCAAGCCAGGTGGTGAATCCGACGGTACCTCCACCACTACTAGCC CACGTTACGATTACCACTGTGGTTTGTCTGACGCTTTGCAACCAGCTCCAG AAGCTGGTACCTGGTTCCAAGCCTACTTCGAACAATTGTTGACTAACGCC AACCCATTGTTCTAACTCGAG (SEQ ID NO: 4) Chryso- ATGGCCAAGAAGTTGTTCATTACCGCTGCCTTAGCTGCCGCAGTGCTTGCT MAKKLFITAALAAAVLAAPVIEE sporium GCACCAGTGATCGAAGAGAGACAAAATTGCGGAGCCGTCTGGACACAGT RQNCGAVWTQCGGNGWQGPTC lucknowense GCGGAGGCAACGGCTGGCAAGGCCCAACATGTTGTGCTTCTGGCTCAACG CASGSTCVAQNEYSQCLPNSQ CBH2b TGCGTGGCACAGAACGAGTGGTATTCCCAGTGCCTTCCAAACTCCCAGGT VTSSTTPSSTSTSQRSTSTSSSTTR GACTTCTTCAACAACCCCCAGCTCAACGTCTACTTCACAGAGATCCACAA SGSSSSSSTTPPPVSSPVTSIPGGA GTACCTCTTCTAGCACAACCAGAAGTGGCTCATCCTCATCTAGCAGTACG TSTASYSGNPFSGVRLFANDYYR ACCCCTCCACCCGTATCAAGTCCTGTCACGAGTATCCCTGGCGGAGCAAC SEVHNLAIPSMTGTLAAKASAV CTCAACAGCCAGTTATTCCGGCAATCCTTTCTCTGGAGTGAGATTATTTGC AEVPSFQWLDRNVTIDTLMVQT AAACGACTATTATAGATCAGAGGTTCACAACCTTGCAATTCCTTCTATGAC LSQVRALNKAGANPPYAAQLVV GGGAACCCTAGCCGCAAAGGCTTCCGCCGTAGCAGAAGTCCCTAGTTTCC YDLPDRDCAAAASNGEFSIANG AATGGCTTGACAGAAACGTTACAATAGATACACTTATGGTACAGACTTTA GAANYRSYIDAIRKHIIEYSDIRII TCTCAGGTTAGAGCTTTGAATAAGGCCGGTGCCAACCCACCTTATGCTGCC LVIEPDSANMVTNMNVAKCS CAATTAGTAGTCTATGACTTGCCAGATAGAGACTGTGCTGCCGCAGCTTCT NAASTYHELTVYALKQLNLPNV AATGGTGAATTTTCCATCGCAAATGGCGGAGCTGCAAACTATAGATCATA AMYLDAGHAGWLGWPANIQPA CATTGATGCAATAAGAAAACACATCATTGAGTATTCTGATATTAGAATAA AELFAGIYNDAGKPAAVRGLAT TCCTTGTGATTGAACCAGACTCCATGGCTAATATGGTTACCAACATGAATG NVANYNAWSIASAPSYTSPNPN TAGCCAAGTGTTCTAACGCAGCTTCCACATACCATGAGCTAACCGTATAT YDEKHYIEAFSPLLNSAGFPARFI GCATTAAAACAACTGAATCTACCTAACGTTGCTATGTACTTAGATGCCGGT VDTGRNGKQPTGQQQWGDWC CATGCCGGATGGTTGGGCTGGCCTGCAAATATCCAACCCGCAGCTGAATT NVKGTGFGVRPTANTGHELVDA GTTCGCTGGAATCTACAACGACGCCGGAAAGCCCGCTGCCGTTAGAGGCT FVWVKPGGESDGTSDTSAARYD TAGCCACAAATGTTGCAAATTACAACGCTTGGTCAATTGCTAGTGCCCCTT YHCGLSDALQPAPEAGQWFQAY CTTATACCTCACCAAATCCTAACTACGATGAGAAACATTACATAGAAGCA FEQLLTNANPPF TTTTCCCCATTGTTAAACTCCGCTGGATTCCCTGCCAGATTCATCGTGGAT (SEQ ID NO: 25) ACCGGTAGAAACGGCAAACAACCAACTGGACAACAACAATGGGGAGATT GGTGTAACGTCAAGGGAACCGGCTTCGGCGTCAGGCCTACGGCAAACACC GGACACGAGCTAGTCGACGCTTTTGTATGGGTTAAGCCAGGTGGCGAAAG TGACGGAACAAGTGACACGAGTGCTGCAAGATACGATTACCACTGTGGTC TGTCCGACGCTTTACAGCCCGCCCCCGAGGCTGGACAATGGTTCCAGGCT TATTTTGAACAATTGTTAACGAACGCAAATCCACCATTCTAA (SEQ ID NO: 5)

Talaromyces ATGCTAAGAAGAGCTTTACTATTGAGCTCTTCTGCTATCTTGGCCGTTAAG MLRRALLLSSSAILAVKAQQAG emersonii GCTCAACAAGCCGGTACCGCTACTGCTGAAAACCACCCTCCATTGACCTG TATAENHPPLTWQECTAPGSCTT cbh1 with GCAAGAATGTACCGCTCCAGGTTCTTGTACCACCCAAAACGGTGCTGTCG QNGAVVLDANWRWVHDVNGY CBD TCTTGGACGCTAACTGGAGATGGGTCCACGACGTCAACGGTTACACTAAC TNCYTGNTWDPTYCPDDETCAQ TGTTACACCGGTAACACCTGGGACCCAACTTACTGTCCAGACGACGAAAC NCALDGADYEGTYGVTSSGSSL TTGCGCTCAAAACTGTGCCTTGGACGGTGCTGACTACGAAGGTACTTACGG KLNFVTGSNVGSRLYLLQDDST TGTTACCTCCTCTGGTTCTTCCTTGAAGTTGAACTTCGTCACTGGTTCTAA YQIFKLLNREFSFDVDVSNLPCG CGTCGGTTCCAGATTGTATTTGTTGCAAGATGACTCCACTTACCAAATCTT LNGALYFVAMDADGGVSKYPN CAAGTTGTTGAACAGAGAATTTTCTTTCGACGTCGATGTGTCCAACTTGCC NKAGAKYGTGYCDSQCPRDLKF CAAGTTGTTGAACAGAGAATTTTCTTTCGACGTCGATGTGTCCAACTTGCC IDGEANVEGWQPSSNNANTGIG TGTTACACCGGTAACACCTGGGACCCAACTTACTGTCCAGACGACGAAAC DHGSCCAEMDVWEANSISNAVT TTGCGCTCAAAACTGTGCCTTGGACGGTGCTGACTACGAAGGTACTTACGG PHPCDTPGQTMCSGDDCGGTYS TGTTACCTCCTCTGGTTCTTCCTTGAAGTTGAACTTCGTCACTGGTTCTAA NDRYAGTCDPDGCDFNPYRMG CGTCGGTTCCAGATTGTATTTGTTGCAAGATGACTCCACTTACCAAATCTT NTSFYGPGKIIDTTKPFTVVTQFL CAAGTTGTTGAACAGAGAATTTTCTTTCGACGTCGATGTGTCCAACTTGCC TDDGTDTGTLSSEIKRFYIQNSNVI TTGTGGTTTGAACGGTGCTCTATACTTCGTTGCTATGGACGCTGATGGTGG PQPNSDISGVTGNSITTEFCTAQK TGTTTCCAAGTACCCAAACAACAAGGCTGGTGCCAAATACGGTACTGGTT QAFGDTDDFSQHGGLAKMGAA ACTGTGACTCTCAATGTCCACGTGACTTGAAGTTTATTGATGGTGAAGCTA MQQGMVLVMSLWDDYAAQML ATGTCGAAGGTTGGCAACCATCTTCTAACAACGCTAACACTGGCATCGGT WLDSDYPTDADPTTPGIARGTCP GACCACGGTTCTTGCTGTGCCGAAATGGACGTTTGGGAAGCCAACTCCAT TDSGVPSDVESQSPNSYVTYSNI TTCCAACGCCGTCACTCCACACCCATGTGACACTCCAGGTCAAACTATGTG KFGPINSTFTASNPPGGNRGTTTT TTCCGGCGATGACTGTGGTGGTACTTACTCTAACGATAGATACGCTGGTAC RRPATTTGSSPGPTQSHYGQCGG CTGTGATCCAGACGGTTGCGACTTCAATCCATACAGAATGGGTAACACTT IGYSGPTVCASGTTCQVLNPYYS CCTTTTACGGTCCAGGCAAGATCATCGACACTACTAAGCCATTCACTGTTG QCL (SEQ ID NO: 26) TCACCCAATTCTTGACCGACGATGGTACTGATACCGGTACTTTGTCCGAAA TCAAGAGATTCTACATCCAAAACTCTAACGTCATCCCACAACCAAATTCC GACATCTCTGGTGTCACTGGTAACTCCATTACCACCGAATTTTGTACCGCC CAAAAGCAAGCTTCGGTGACACCGACGACTTCTCTCAACACGGTGGTTT GGCTAAGATGGGTGCTGCTATGCAACAAGGTATGGTTTTGGTCATGTCTTT GTGGGACGACTACGCTGCTCAAATGTTGTGGTTGGACTCCGATTACCCAA CCGATGCCGACCCAACCACCCCTGGTATCGCTAGAGGTACCTGTCCAACT GACTCTGGTGTTCCATCTGACGTCGAATCCCAATCTCCAAACTCCTACGTC ACTTACTCCAACATTAAATTCGGTCCAATCAACTCCACTTTCACTGCTTCT AACCCTCCAGGTGGTAACAGAGGTACTACCACTACTCGTAGGCCAGCTAC TACAACTGGTTCTTCCCCAGGCCCAACCCAATCCCACTACGGTCAATGTGG TGGTATCGGTTACTCTGGTCCAACCGTCTGTGCTTCTGGTACTACCTGTCA AGTTTTAAACCCATACTACTCTCAATGTTTGTAG (SEQ ID NO: 6) Trichoderma ATGGTCTCCTTCACCTCCCTGCTGGCCGGCGTTGCCGCTATCTCTGGTGTC ACCESSION NO: CAA49596 reesei CBH1 CTAGCAGCCCCTGCCGCAGAAGTTGAACCTGTCGCAGTTGAGAAACGTGA MVSFTSLLAGVAAISGVLAAPA GGCCGAAGCAGAAGCTCAATCCGCTTGTACCCTACAATCCGAAACTCACC AEVEPVAVEKREAEAEAQSACT CACCATTGACCTGGCAAAAGTGTTCTAGCGGTGGAACTTGTACTCAACAA LQSETHPPLTWQKCSSGGTCTQ ACTGGTTCTGTTGTTATCGACGCTAACTGGAGATGGACACACGCCACTAA QTGSVVIDANWRWTHATNSSTN CTCTTCTACCAACTGTTACGACGGTAACACTTGGTCTTCCACTTTATGTCC CYDGNTWSSTLCPDNETCAKNC AGATAACGAAACTTGTGCTAAGAATTGCTGTTTGGACGGTGCCGCCTACGC CLDGAAYASTYGVTTSGNSLSIG TTCTACCTACGGTGTTACCACCTCCGGTAACTCCTTGTCTATTGGTTTCGT FVTQSAQKNVGARLYMASDTT CACTCAATCCGCTCAAAAGAACGTTGGTGCTAGATTGTACTTGATGGCTTC YQEFTLLGNEFSFDVDVSQLPCG TGACACTACTTATCAAGAATTTACTTTGTTGGGTAACGAATTTTCTTTCGA LNGALYFVSMDADGGVSKYPTN TGTTGACGTTTCCCAATTGCCATGTGGCTTGAACGGTGCTTTGTACTTTGT TAGAKYGTGYCDSQCDSQCPRDLKFI CTCTATGGATGCTGACGGTGGTGTTTCTAAGTACCCAACTAACACTGCCGG NGQANVEGWEPSSNNANTGIGG TGCTAAGTACGGTACTGGTTACTGTGATTCTCAATGTCCACGTGACTTGAA HGSCCSEMDIWEANSISEALTPH GTTCATTAACGGTCAAGCCAACGTCGAAGGTTGGGAACCATCCTCCAACA PCTTVGQEICEGDGCGGTYSDN ACGCTAACACCGGTATCGGTGGTCACGGTTCCTGTTGTTCCGAAATGGAC RYGGTCDPDGCDWNPYRLGNTS ATCTGGGAAGCTAACAGTATTTCTGAAGCTTTGACACCACACCCATGCAC FYGPGSSFTLDTTKKLTVVTQFE CACTGTCGGTCAAGAAATTTGTGAAGGTGATGGATGTGGTGGAACCTACT TSGAINRYYVQNGVTFQQPNAE CTGATAACAGATACGGTGGTACTTGTGACCCAGACGGTTGTGACTGGAACC LGSYSGNELNDDYCTAEEAEFG CATACAGATTGGGTAACACTTCTTTCTATGGTCCAGGTTCTTCTTTCACCT GSSFSDKGGLTQFKKATSGGMV TGGATACCACCAAGAAGTTGACTGTTGTTACCCAATTCGAAACTTCTGGTG LVMSLWDDYYANMLWLDSTYP CTATCAACAGATACTACGTTCAAAACGGTGTCACCTTCCAACAACCAAAC TNETSSTPGAVRGSCSTSSGVPA GCTGAATTGGGTTCTTACTCTGGTAATGAATTGAACGACGACTACTGTACC QVESQSPNAKVTFSNIKFGPIGST GCTGAAGAAGCTGAATTTGGTGGTTCCTCTTTCTCCGACAAGGGTGGTTTG GNPSGGNPPGGNRGTTTTRRPAT ACCCAATTCAAGAAGGCTACCTCCGGTGGTATGGTTTTGGTTATGTCCTTG TTGSSPGPTQSHYGQCGGIGYSG TGGGATGATTACTACGCAAACATGTTATGGTTAGACAGTACTTACCCAAC PTVCASGTTCQVLNPYYSQCL TAACGAAACCTCCTCTACTCCAGGTGCTGTCAGAGGTTCCTGTTCTACCTC (SEQ ID NO: 27) TTCTGGTGTTCCAGCTCAAGTTGAATCTCAATCTCCAAACGCTAAGGTCAC [SECRETIONAL SIGNAL: 1-33 TTTCTCCAACATCAAGTTCGGTCCAATCGGTTCCACTGGTAATCCATCTGG CATALYTIC DOMAIN: 41-465 TGGAAACCCTCCAGGTGGTAACAGAGGTACTACCACTACTCGTAGGCCAG CELLULOSE-BINDING DOMAIN: CTACTACAACTGGTTCTTCCCCAGGCCCAACCCAATCCCACTACGGTCAAT 503-535] GTGGTGGTATCGGTTACTCTGGTCCAACCGTCTGTGCTTCTGGTACTACCT GTCAAGTTTTAAACCCATACTACTCTCAATGTTTGTAA (SEQ ID NO: 7) Trichoderma ATGGTCTCCTTCACCTCCCTGCTGGCCGGCGTTGCCGCTATCTCTGGTGTC ACCESSION NO: reesie CBH2 CTAGCAGCCCCTGCCGCAGAAGTTGAACCTGTCGCAGTTGAGAAACGTGA AAA72922AAA34210 GGCCGAAGCAGAAGCTGTCCCATTAGAAGAAAGACAAGCCTGCTCCTCTGT MIVGILTTLATLATLAASVPLEE TTGGGGTCAATGTGGTGGTCAAAACTGGTCTGGTCCAACTTGTTGTGCTTC RQACSSVWGQCGGQNWSGPTC CGGTTCTACCTGTGTTTACTCCAACGACTACTATTCCCAATGTTTGCCAGG CASGSTCVYSNDYYSQCLPGAA TGCTGCTTCCTCTTCCTCTTCAACTAGAGCTGCTTCTACAACTTCTAGGGT SSSSTRAASTTSRVSPTTSRSSS CTCCCCAACCACTTCCAGATCCTCTTCTGCTACTCCACCACCAGGTTCTAC ATPPPGSTTTRVPPVGSGTATYS TACCACTAGAGTTCCACCAGTCGGTTCCGGTACTGCTACTTACTCTGGTAA GNPFVGVTPWANAYYASEVSSL CCCTTTCGTCGGTGTTACTCCATGGGCTAACGCTTACTACGCTTCTGAAGT AIPSLTGAMATAAAAVAKVPSF TTCTTCTTTGGCTATCCCATCTTTGACTGGTGCTATGGCTACCGCTGCTGC MWLDTLDKTPLMEQTLADIRTA TGCTGTCGCCAAAGTTCCATCCTTCATGTGGTTGGACACCTTGGACAAAAC NKNGGNYAGQFVVYDLPDRDC TCCATTAATGGAACAAACCTTGGCAGACATAAGGACTGCTAACAAGAACG AALASNGEYSIADGGVAKYKNY GCGGTAACTACGCTGGTCAATTTGTTGTGTACGACTTGCCAGACAGAGAC IDTIRQIVVEYSDIRTLLVIEPDSL TGTGCTGCTTTGGCTTCCAACGGTGAATACTCCATCGCTGACGGTGGTGTC ANLVTNLGTPKCANAQSAYLEC GCCAAGTACAAGAACTACATTGATACCATTAGACAAATCGTTGTCGAATA INYAVTQLNLPNVAMYLDAGHA CTCTGACATCAGAACCTTGTAAGTCATCGAACCAGATTCTTTAGCCAATTT GWLGWPANQDPAAQLFANVYK AGTCACCAACTTGGGTACTCCAAAGTGTGCTAACGCTCAATCTGCCTACTT NASSPRALRGLATNVANYNGW AGAATGTATCAATTATGCAGTTACCCAATTGAACTTGCCAAACGTTGCTAT NITSPPSYTQGNAVYNEKLYIHAI GTACTTGGACGCTGGTCACGCCGGTTGGTTGGGTTGGCCAGCTAACCAAG GRLLANHGWSNAFFITDQGRSG ACCCAGCCGCTCAATTATTCGCCAACGTTTACAAGAATGCCTCTTCTCCTA KQPTGQQQWGDWCNVIGTGFGI GAGCCTTGCGTGGTTTGGCTACTAACGTCGCTAACTACAACGGTTGGAAC RPSANTGDSLLDSFVWVKPGGE ATCACTTCTCCACCATCTTACACCCAAGGTAACGCTGTTTACAACGAAAA CDGTSDSSAPRFDSHCALPDALQ GTTGTACATTCACGCTATCGGTCCATTATTGGCTAACCATGGTTGGTCTAA PAAQAGAWFQAYFVQLLTNAN CGCCTTCTTCATCACCGACCAAGGTAGATCCGGTAAACAACCAACTGGTC PSFL (SEQ ID NO: 28) AACAACAATGGGGTGATTGGTGTAACGTCATCGGTACTGGTTTCGGTATC AGACCATCCGCTAACACTGGTGATTCCTTGTTGGATTCCTTCGTCTGGGT AAGCCAGGTGGTGAATGTGATGGCACCTCTGATTCCTCTGCTCCAAGATTC GATTCCCACTGCGCCTTGCCAGACGCTTTGCAACCAGCCCCACAAGCTGG TGCATGGTTCCAAGCTTACTTTGTCCAATTGTTGACCAACGCTAACCCATC TTTCTTGTAA (SEQ ID NO: 8) Chaetomium TTAATTAAACAATGATGTACAAGAAATTTGCAGCCCTAGCTGCTTTAGTTG AM711862 thermo- CAGGAGCTTCCGCTCAACAGGCATGTTCATTGACTGCCGAAAATCATCCA MMYKKFAALAALVAGASAQQA philum CBH1 TCCTTAACGTGGAAGAGATGCACGTCAGGAGGTTCATGCTCCACTGTAAA CSLTAENHPSLTWKRCTSGGSCS CGGAGCTGTCACAATAGATGCAAATTGGAGATGGACCCACACTGTGTCCG TVNGAVTIDANWRWTH GTAGTACAAACTGCTACACCGGTAATCAATGGGATACGTCTTTGTGTACA TVSGSTNCYTGNQWDTSLCTDG GATGGAAAGTCATGCGCTCAGACCTGTTGCGTGGATGGAGCAGACTACTC KSACAQTCCVDGADYSSTYGITTS TTCTACTTACGGAATCACGACATCAGGTGACAGTCTTAATTTGAAATTCGT GDSLNLKFVTKHQYG AACCAAGCACCAGTACGGAACAAATGTAGGCTCCAGAGTGTACTTAATGG TNVGSRVYLMENDTKYQMFELL AGAACGATACCAAATATCAAATGTTCGAGTTATTAGGCAATGAGTTTACC GNEFTFDVDVSNLGCGLNGALY TTTGACGTAGACGTTAGCAATTTGGGTTGCGGATTAAACGGCGCCCTTAC FVSMDADGGMSKYSGN TTCGTGTCTATGGATGCTGACGGAGGTATGTCAAAGTATTCTGGTAACAA KAGAKYGTGYCDAQCPRDLKFI AGCCGGAGCAAAGTACGGTACAGGTTATTGTGACGCTCAGTGCCCTAGAG NGEANVGNWTPSTNDANAGFG ATTTGAAGTTTATCAACGGAGAAGCCAACGTTGGTAACTGGACGCCAAGT RYGSCCSEMDVWEANNM ACTAACGACGCAAACGCTGGATTCGGCAGATACGGTAGTTGTTGCTCAGA ATAFTPHPCTTVGQSRCEADTCG AATGGACGTGTGGGAGGCCAATAACATGGCAACCGCTTTTACTCCTCACC GTYSSDRYAGVCDPDGCDFNAY CATGTACAACTGTTGGACAATCTAGATGTGAAGCCGACACGTGCGGTGGC RQGDKTFYGKGMTVD ACCTACAGTAGCGATAGGTATGCAGGAGTATGTGATCCTGACGGTTGCGA TNKKMTVVTQFHKNSAGVLSEI TTTCAATGCTTATAGACAAGGAGACAAAACGTTTTATGGTAAAGGTATGA KRFYVQDGKIIANAESKIPGNPG CCGTCGATACTAACAAGAAGATGACTGTGGTTACCCAGTTCCACAAGAAC NSITQEYCDAQKVAF TCAGCTGGAGTATTGTCTGAAATTAAAAGATTCTACGTCCAGGATGGAAA SNTDDFNRKGGMAQMSKALAG GATTATTGCTAATGCCGAGAGTAAGATACCAGGTAACCCTGGAAATAGTA PMVLVMSVWDDHYANMLWLD TCACACAGGAATACTGTGACGCTCAGAAGGTAGCTTTTAGCAACACCGAT STYPIDQAGAPGAERGACP GACTTCAATAGAAAGGGTGGAATGGCTCAAATGAGTAAGGCTTTAGCCGG TTSGVPAEIEAQVPNSNVIFSNIR TCCAATGGTGTTGGTGATGTCTGTTTGGGATGATCACTATGCAAACATGCT FGPIGSTVPGLDGSNPGNPTTTV TTGGCTTGACAGCACCTATCCTATCGACCAAGCCGGAGCCCCAGGTGCTG VPPASTSTSRPTS AAAGGGGTGCATGTCCAACCACGAGTGGTGTGCCCGCCGAGATTGAAGCT STSSPVSTPTGQPGGCTTQKWGQ CAAGTGCCTAATAGTAACGTTATCTTTTCCAATATAAGATTCGGACCAATC CGGIGYTGCTNCVAGTTCTQLN GGATCCACTGTTCCAGGTTTGGATGGATCTAATCCTGGCAACCCAACAAC PWYSQCL (SEQ ID NO: 29) CACGGTAGTCCCTCCAGCTTCAACTTCCACAAGTAGACCAACAAGTTCAA CGTCCAGTCCAGTGTCTACTCCTACCGGACAACCAGGAGGCTGTACCACT CAGAAATGGGGTCAATGCGGTGGAATTGGCTATACAGGTTGTACGAATTG CGTTCAGGAACCACTTGTACACAGTTAAACCCTTGGTACTCACAATGCCT ATAAGGCGCGCC (SEQ ID NO: 9) Acremonium ATGTATACCAAATTTGCTGCATTGGCCGCTTTAGTTGCAACAGTAAGAGGT MYTKFAALAALVATVRGQAAC thermo- CAAGCCGCTTGTTCTCTAACCGCAGAAACTCACCCATCTCTACAATGGCA SLTAETHPSLQWQKCTAPGSCTT philum CBH1 GAAATGCACAGCCCCTGGATCTTGTACAACTGTCTCCGGCCAAGTCACCA VSGQVTIDANWRWLHQTNSSTN TTGACGCTAATTGGAGATGGCTTCACCAAACTAACTCTTCAACGAATTGTT CYTGNEWDTSICSSDTDCATKC ATACCGGTAACGAATGGGATACTTCCATATGTTCATCCGATACAGACTGC CLDGADYTGTYGVTASGNSLNL GCAACGAAATGTTGTTTAGATGGAGCAGACTATACGGGAACTTATGGTGT KFVTQGPYSKNIGSRMYLMESES TACAGCCTCAGGTAATTCCCTAAACCTTAAGTTCGTAACTCAAGGACCAT KYQGFTLLGQEFTFDVDVSNLG ATAGTAAGAATATCGGCTCTAGAATGTACTTGATGGAAAGTGAGAGCAAA CGLNGALYFVSMDLDGGVSKYT TATCAGGGTTTTACGTTATTGGGACAAGAGTTTACATTTGATGTTGATGTG TNKAGAKYGTGYCDSQCPRDLK AGTAACTTAGGTTGCGGCCTAAACGGCGCCTTGTACTTCGTTTCTATGGAT FINGQANIDGWQPSSNDANAGL CTTGATGGAGGTGTATCAAAATACACGACCAACAAGGCTGGAGCCAAATA GNHGSCCSEMDIWEAMKVSAAY TGGTACGGGATATTGTGACAGCCAATGCCCTAGAGACTTAAAGTTCATTA TPHPCTTIGQTMCTGDDCGGTYS ACGGTCAGGCAAATATTGACGGCTGGCAACCAAGCAGTAACGACGCTAAT SDRYAGICDPDGCDFNSYRMGD GCCGGACTAGGTAACCATGGCTCATGTTGTTCCGAAATGGATATCTGGGA

TSFYGPGKTVDTGSKFTVVTQFL AGCCAATAAGGTGTCCGCTGCCTACACCCCCCATCCATGCACGACAATCG TGSDGNLSEIKRFYVQNGKVIPN GTCAGACAATGTGTACCGGTGATGACTGTGGAGGCACATACTCAAGTGAT SESKIAGVSGNSITTDFCTAQKT AGGTACGCCGGTATATGTGATCCTGACGGTTGCGATTTCAACTCTTATAGA AFGDTNVFEERGGLAQMGKAL ATGGGAGATACATCCTTTTACGGCCCCGGTAAAACAGTTGATACGGGTAG AEPMVLVLSVWDDHAVNMLWL TAAGTTCACTGTTGTTACTCAGTTCTTAACAGGTTCAGACGGCAATCTTAG DSTYPTDSTKPGAARGDCPITSG TGAAATCAAAAGATTCTACGTTCAGAATGGAAAAGTCATTCCTAATTCCG VPADVESQAPNSNVIYSNIRFGPI AGAGTAAGATTGCTGGTGTGTCTGGTAACAGTATCACGACCGACTTCTGT NSTYTGTPSGGNPPGGGTTTTTT ACCGCCCAAAAGACTGCCTTTGGAGATACGAATGTTTTCGAGGAAAGGGG TTTSKPSGPTTTTNPSGPQQTHW CGGTCTTGCTCAAATGGGCAAGGCTTTGGCCGAACCAATGGTATTAGTCC GQCGGQGWTGPTVCQSPYTCK TATCCGTTTGGGATGATCATGCAGTGAATATGCTTTGGCTTGATAGCACCT YSNDWYSQCL ACCCTACTGACAGCACCAAGCCAGGAGCTGCCAGAGGTGACTGTCCTATC (SEQ ID NO: 46) ACAAGTGGCGTTCCAGCAGATGTAGAGAGCCAAGCTCCAAACTCCAATGT GATCTATTCTAACATCAGATTTGGCCCCATTAATAGTACCTATACAGGAAC GCCCTCTGGTGGTAACCCTCCAGGCGGAGGCACCACAACTACCACGACCA CAACGACTTCAAAGCCTTCTGGCCCTACGACAACTACCAATCCTTCCGGA CCACAGCAAACTCACTGGGGTCAGTGTGGAGGCCAAGGATGGACGGGTC CTACCGTGTGTCAATCACCTTACACATGCAAATACAGTAATGACTGGTACT CTCAGTGTTTATAA (SEQ ID NO: 45) Endoglucanases Coptotermes ATGAGATTTCCTTCCATATTCACCGCTGTTTTGTTCGCAGCCTCAAGTGCT MRFPSIFTAVLFAASSALAECTK lacteus EG TTAGCAGAATGTACTAAGGGTGGATGTACTAACAAGAATGGATACATAGTT GGCTNKNGYIVHDKHVGDIQNR CATGATAAGCACGTCGGTGACATCCAGAATAGAGACACTTTGGACCCTCC DTLDPPDLDYEKDVGVTVSGGT AGACTTAGATTATGAAAAGGACGTGGGAGTAACCGTGTCCGGTGGAACCC LSQRLVSTWNGKKVVGSRLYIV TTAGTCAAAGATTAGTCTCAACTTGGAACGGTAAGAAAGTCGTGGGAAGT DEADEKYQLFTFVGKEFTYTVD AGATTGTATATTGTGGACGAAGCCGACGAGAAATATCAATTATTCACATTT MSQIQCGINAALYTVEMPAAGK GTCGGTAAGGAGTTCACCTATACCGTTGATATGTCCCAGATCCAATGTGGA TPGGVKYGYGYCDANCVDGDC ATCAATGCCGCATTATACACAGTGGAAATGCCTGCCGCTGGAAAGACCCC CMEFDIQEASNKAIVTTHSCQS TGGAGGTGTTAAGTATGGATATGGATATTGTGATGCCAACTGCGTGGATG QTSGCDRTSGCGYNPYRDSGDKA GAGATTGTTGTATGGAGTTCGATATCCAAGAAGCTTCTAACAAGGCAATC FWGTTINVNQPVTIVTQFIGSGSS GTTTACACCACCCATTCCTGTCAAAGTCAAACTTCAGGTTGCGATACCTCA LTEVKRLCVQGGKTFPPAKSLT GGATGCGGTTACAACCCTTACAGAGACAGTGGTGACAAGGCATTCTGGGG DSYCNANDYRSLRTMGASMAR AACAACTATAAACGTAAACCAGCCTGTGACAATTGTAACACAGTTTATCG GHVVVFSLWDSNGMSWMDGG GTTCTGGTAGTTCCTTAACTGAAGTCAAAAGATTGTGCGTGCAAGGTGGA NAGPCTSYNIESLESSQPNLKVT AAGACCTTCCCTCCAGCCAAATCATTAACCGACAGTTATTGTAATGCCAAC WSNVKYGEIDSPY GACTATAGAAGTTTGAGAACTATGGGTGCATCCATGGCTAGAGGACACGT (SEQ ID NO: 30) TGTTGTGTTTTCTTTGTGGGATTCTAATGGTATGAGTTGGATGGATGGAGG TAACGCCGGTCCTTGTACCTCATATAATATTGAATCTTTGGAATCCAGTCA GCCAAACTTAAAGGTCACATGGTCAAACGTGAAATACGGAGAGATCGATT GCCAAACTTAAAGGTCACATGGTCAAACGTGAAATACGGAGAGATCGATT CTCCTTATTAA (SEQ ID NO: 10) Coptotermes ATGAGATTCCCTTCCATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCT BAB40697 formosanus TTAGCAGCCTATGACTACAAGACAGTATTGAAGAACTCCTTGTTGTTCTAC MRFPSIFTAVLFAASSALAAYDY EG GAAGCTCAAAGAAGTGGAAAATTGCCTGCAGACCAGAAGGTGACCTGGAG KTVLKNSLLFYEAQRSGKLPAD AAAAGATTCCGCATTAAACGACAAGGGACAGAAGGGAGAGGACTTAACT QKVTWRKDSALNDKGQKGEDL GGAGGTTATTACGACGCCGGAGACTTTGTGAAGTTCGGTTTTCCAATGGCA TGGYYDAGDFVKFGFPMAYTV TACACAGTTACCGTGTTGGCCTGGGGTTTAGTCGATTATGAATCTGCTTAC TVLAWGLVDYESAYSTAGALD AGTACTGCGGGTGCCTTGGATGATGGTAGAAAGGCCTTGAAATGGGGTAC DGRKALKWGTDYFLKAHTAAN AGATTATTTCTTGAAAGCACATACCGCTGCCAATGAGTTTTACGGACAGGT EFYGQVGQGDVDHAYWGRPED GGGTCAGGGAGATGTGGATCATGCTTACTGGGGACGTCCTGAGGACATGA MTMSRPAYKIDTSKPGSDLAAE CTATGTCTAGACCAGCTTACAAGATCGATACATCAAAACCTGGTAGTGACT TAAALAATAIAYKSADSTYSNN TAGCTGCAGAAACAGCAGCCGCTTTAGCAGCAACCGCAATAGCTTACAAG LITHAKQLFDFANNYRGKYSDSI TCAGCCGATTCTACCTACAGTAACAACTTAATTACTCATGCAAAGCAGTTG TDAKNFYASGDYKDELVWAAA TTCGATTTTGCAAACAATTATAGAGGAAAGTACTCTGATAGTATTACCGAT WLYRATNDNTYLTKAESLYNEF GCCAAGAATTTCTATGCATCCGGTGATTATAAGGACGAATTAGTATGGGCT GLGSWNGAFNWDNKISGVQVL GCAGCCTGGTTGTATAGAGCTACAAATGATAACACTTACTTAACCAAAGC LAKLTSKQAYKDKVQGYVDYL CGAATCATTGTATAATGAATTTGGTTTAGGATCTTGGAACGGTGCATTCAA VSSQKKTPKGLVYIDQWGTLRH TTGGGATAACAAGATATCCGGAGTTCAGGTCTTATTAGCCAAATTGACATC AANSALIALQAADGINAASYR CAAACAAGCATACAAAGATAAAGTTCAGGGTTATGTTGATTACTTAGTCTC QYAKKQIDYALGDGGRSYVVG CTCTCAAAAGAAAACTCCAAAGGGATTGGTCTATATTGACCAATGGGGAA FGTNPPVRPHHRSSSCPDAPAAC CCTTAAGACACGCAGCTAATAGTGCCTTGATCGCTTTACAGGCCGCTGATT DWNTYNSAGPNAHVLTGALVG TGGGTATAAACGCTGCTAGTTATAGACAATACGCAAAGAAGCAAATTGATT GPDSNDSYTDSRSDYISNEVATD ATGCCTTAGGTGACGGAGGTCGTTCTTACGTGGTCGGATTCGGAACTAACC YNAGFQSAVAGLLKAGV CTCCAGTAAGACCTCATCATAGATCCAGTTCCTGTCCTGACGCACCACGCC (SEQ ID NO: 31) GCTTGCGACTGGAATACTTACAACTCTGCCGGACCAAATGCCCACGTCTTG ACCGGAGCCTTAGTAGGTGGACCAGATTCCAACGATAGTTACACAGATTC ACGTTCTGATTATATCAGTAACGAAGTCGCTACTGATTACAATGCCGGTTT CCAATCTGCAGTTGCTGGTTTGTTGAAAGCCGGAGTATAA (SEQ ID NO: 11) Nasutitermes ATGAGATTTCCATCTATTTTCACTGCCGTCTTATTTGCAGCCTCACAGTGC MRFPSIFTAVLFAASSALAAYDY takasa- ATAAAAGATTCAGCCTTGAATGATCAGGGAGATCAAGGTCAAGACTTAACC KQVLRDSLLFYEAQRSGRLPAD goensis EG GGAGGTTATTTTGACGCCGGTGATTTTGTGAAATTTGGTTTCCCAATGGCA QKVTWRKDSALNDQGDQGQDL TATACTGCTACCGTCTTGGCCTGGGGTTTAATCGATTTTGAGGCAGGATAC TGGYFDAGDFVKFGFPMAYTAT AGTTCCGCTGGTGCCTTGGATGACGGTAGAAAAGCAGTAAAGTGGGCAACT VLAWGLIDFEAGYSSAGALDDG GATTACTTTATAAAGGCCCACACTTCACAGAATGAGTTTTACGGACAAGTC RKAVKWATDYFIKAHTSQNEFY GGTCAGGGTGACGCTGATCACGCTTTCTGGGGACGTCCTGAAGATATGAC GQVGQGDADHAFWGRPEDMT CATGGCTAGACCAGCCTACAAGATTGACACCAGCAGACCAGGTAGTGACT MARPAYKIDTSRPGSDLAGETA TAGCGGGTGAAACCGCAGCGGCATTGGCAGCTGCCAGTATCGTGTTTAGA AALAAASIVFRNVDGTYSNNLL AATGTTGATGGTACATACTCTAACAACTTACTTACTCATGCCAGACAATTA THARQLFDFANNYRGKYSDSIT TTTGACTTTGCAAATAACTACAGAGGAAAATACTCAGATTCCATAACCGA DARNFYASADYRDELVWAAAW CGCTAGAAACTTTTACGCCAGTGCAGATTACCGTGACGAATTGGTTTGGGC LYRATNDNTYLNTAESLYDEFG TGCCGCATGGTTGTACAGAGCTACAAATGACAACACTTACTTGAATACCG LQNWGGGLNWDSKVSGVQVLL CAGAATCCTTGTATGATGAATTTGGATTGCAGAACTGGGGTGGAGGGTTA AKLTNKQAYKDTVQSYVNYLIN AACTGGGATTCAAAGGTGTCTGGTGTCCAGGTCTTGTTAGCAAAATTGACC NQQKTPKGLLYIDMWGTLRHA AACAAACAGGCTTACAAAGATACTGTGCAGTCTTACGTGAATTACCTGATT ANAAFIMLEAAELGLSASSYRQF AATAACCAGCAAAAGACCCCAAAAGGATTGTTATACATTGATATGTGGGG AQTQIDYALGDGGRSFVCGFGS TACATTGAGACACGCCGCAAATGCTGCATTCATCATGTTGGAAGCTGCCG NPPTRPHHRSSSCPPAPATCDWN AGTTGGGTTTATCCGCATCATCTTACAGACAGTTTGCTCAAACTCAGATCG TFNSPDPNYHVLSGALVGGPHDQ ACTACGCTTTGGGTGACGGTGGAAGAAGTTTCGTCTGTGGTTTTGGTTCAA NDNYVDDRSDYVHNEVATDYN ACCCTCCTACAAGACCACATCATCGTTCTTCCAGTTGCCCGCCTGCCCCAG AGFQSALAALVALGY CAACTTGTGACTGGAATACATTCAACTCACCTGACCCAAATTACCACGTGT (SEQ ID NO: 32) TATCTGGAGCTTTGGTAGGAGGACCAGATCAAAACGATAATTATGTGGAT GATAGATCCGACTACGTCCATAACGAAGTGGCAACCGACTACAACGCCGG ATTTCAGAGTGCTTTGGCAGCCTTAGTTGCTTTGGGTTATTAA (SEQ ID NO: 12) Coptotermes ATGAGATTCCCTAGTATTTTCACTGCCGTCTTATTTGCAGCCAGTTCTGCT MRFPSIFTAVLFAASSALAAYDY acinaciformis TTAGCCGCATATGATTATACCACAGTTTTGAAAAGTTCCTTATTGTTCTAC TTVLKSSLLFYEAQRSGKLPADQ EG GAAGCTCAAAGATCCGGTAAGTTGCCAGCCGACCAGAAGGTCACTTGGAGA KVTWRKDSALDDKGNNGEDLT AAAGATTCAGCATTAGACGATAAAGGAAATAATGGAGAGGACTTAACAGG GGYYDAGFVKFGFPLAYTATV AGGTTATTATGACGCTGGTGATTTTGTGAAGTTTGGTTTTCCTTTAGCATA LAWGLVDYEAGYSSAGATDDG CACCGCTACTGTTTTAGCCTGGGGTTTGGTGGACTATGAAGCGGGTTACTC RKAVWATDYLLKAHTAAEL ATCCGCTGGAGCCACAGATGACGGTAGAAAGGCAGTGAAATGGGCAACC YGQVGDGDADHAYWGRPEDM GACTATTTGTTGAAGGCACATACTGCCGCTACCGAGTTATACGGCAGGTC TMARPAYKIDASPGSDLAGET GGGGACGGTGACGCCGATCACGCATATTGGGGACGTCCTGAAGATATGAC AAALAAASIVFKGVDSSYSDNL TATGGCTAGACCAGCATACAAGATCGACGCTAGCAGACCAGGATCTGACT LAHAKQLFDFADNYRGKYSDSI TAGCGGGTGAAACCGCTGCCGCTTTAGCCGCTGCATCCATAGTTTTCAAAG TQASNFYASGDYKDELVWAAT GTGTAGATTCTTCATATTCTGACAACTTGTTAGCTCACGCTAAACAGTTAT WLYRATNDTYLTKAESLYNEF TTGATTTCGCTGACAATTATAGAGGAAAATACAGTGATTCCATAACACAA GLGNWNGAFNWDNKVSGVQV GCTTCAAACTTTTACGCCTCCGGAGATTACAAAGACGAGTTAGTCTGGGCT LLAKLTSKQAYKDTVQGYVDY GCCACTTGGTTGTACAGAGCAACCAACGATAATACATATTTGACCAAAGC LINNQQKTPKGLLYIDQWTLR AGAATCCTTGTACAACGAGTTCGGATTAGGAAACTGGAACGGAGCCTTTA HAANAALIILQAADLGISADSYR ATTGGGACAACAAGGTGTCCGGTGTTCAGGTGTTGTTAGCCAAATTGACCT QFAKKQIDYALGDGGRSYVVGF CCAAGCAGGCTTATAAAGACACCGTTCAAGGATACGTCGATTATTTGATTA GDNPPTHPHHRSSSCPDAPAVC ACAATCAGCAAAAGACCCCAAAGGGTTTGTTATACATAGACCAATGGGGG DWNTFNSPDPNFHVLTGALVGG ACCTTGAGACACGCAGCTAATGCTGCCTTAATAATCTTACAGGCTGCTGAT PDQNDNYVDDRSDYVSNEVAT TTGGGTATTTCTGCCGACAGTTATAGACAATTCGCAAAGAAGCAAATAGA DYNAGFSAVAALVTLGV TTACGCTTTAGGTGACGGAGGTAGATCATATGTAGTTGGTTTTGGAGACAA (SEQ ID NO: 33) TCCTCCAACACATCCTCATCACCGTTCTTCCTCATGCCCTGACGCCCCAGC AGTATGCGATTGGAATACTTTCAATTCACCTGATCCAAACTTTCATGTCTT AACCGGAGCTTTAGTGGGAGGTCCTGATCAGAACGATAACTACGTTGATG ATCGTTCTGACTACGTGTCCAACGAGGTTGCAACCGACTATAATGCAGGAT TCCAAAGTGCTGTGGCCGCTTTAGTTACTTTAGGAGTTTAA (SEQ ID NO: 13) Mastotermes ATGAGATTCCCAAGTATATTTACTGCTGTTTTGTTCGCAGCCAGTTCTGCT MRFPSIFTAVLFAASSALAAYDY darwinensis TTAGCAGCCTATGATTACAATGACGTATTAACCAAAAGTTTGTTGTTCTAC NDVLTKSLLFYEAQRSGKLPSD EG GAAGCTCAAAGATCCGGTAAGTTACCTTCTGATCAGAAAGTCACCTGGAGA QKVTWRKDSALNDKGQNGEDL AAAGATTCAGCATTAAACGATAAGGGACAAAATGGTGAGGACTTAACTGG TGGYYDAGDYVKFGFPMAYTA TGGATATTATGACGCCGGTGATTACGTGAAGTTTGGTTTTCCAATGGCATA TVLAWGLVDHPAGYSSAGVLD TACTGCTACCGTTTTGGCTTGGGGTTTAGTGGACCATCCTGCCGGATACAG DGRKAVKWVTDYLIKAHVSKN TTCTGCGGGTGTCTTGGATGATGGTAGCCCCGCTGTGAAGTGGGTTACCG ELYGQVGDGDADHAYWGRPED ATTACTTAATCAAAGCCCACGTATCAAAGAACGAATTATACGGACAGGTC MTMARPAYKIDTSRPGSDLAGE GGTGACGGTGACGCAGATCACGCTTATTGGGGACGTCCAGAGGATATGAC TAAALAAASIVFKSTDNYANT AATGGCAAGACCAGCATACAAAATAGACACTTCAAGACCAGGTTCCGACT LLTHAKQLFDFANNYRGKYSDS TAGCGGGTGAAACCGCAGCGGCATTGGCTGCTGCATCTATTGTGTTTAAGT ITQASNFYSSSDYKDELVWAAV CAACAGATTCTAATTACGCCAACACCTTATTGACCCACGCAAAACAATTAT WLYRATNDQTYLTTAEKLYSDL TCGACTTTGCCAATAACTATAGAGGTAAGTATAGTGATTCCATAACACAG GLQSWNGGFTWDTKISGVEVLL GCATCTAATTTCTACAGTAGTTCCGACTATAAAGATGAATTGGTTTGGGCA AKITGKQAYKDKVKGYCDYISG GCTGTATGGTTGTACAGAGCCACTAACGATCAGACCTATTTGACAACTGCA SQQKTPKGLVYIDKWGSLRMA GAGAAGTTATACTCAGACTTGGGATTACAGTCCTGGAACGGAGGTTTCAC ANAAYICAVAADVGISSTAYRQ ATGGGACACCAAAATTAGTGGAGTAGAAGTGTTATTGGCTAAGATTACTG FAKTQINYILGDAGRSFVVGYG GTAAACAGGCATATAAGGACAAAGTAAAGGGATATTGTGATTATATCTCA NNPPTHPHHRSSSCPDAPATCD GGATCTCAGCAGAAAACACCTAAAGGATTAGTTTACATAGATAAGTGGGG WNNYNSANPNPHVLYGALVGG TTCCTTAAGAATGGCCGCAAACGCCGCATATATTTGCGCTGTAGCCGCAGA PDSNDNYQDLRSDYVANEVAT CGTCGGAATCAGTTCAACAGCTTACAGACAGTTCGCCAAAACACAGATTA DYNAAFQSLLALIVDLGL ATTACATATTGGGTGATGCCGGACGTTCTTTTGTGGTTGGTTACGGAAACA (SEQ ID NO: 34) ACCCACCTACACACCCACATCACAGATCCAGTTCATGTCCTGACGCCCCAG CAACATGCGATTGGAATAACTACAACAGTGCTAACCCTAATCCACATGTTT TATACGGTGCATTAGTTGGTGGACCAGATTCCAACGATAATTATCAAGACT TAAGATCAGATTATGTCGCCAACGAAGTGGCAACAGACTACAATGCAGCC TTCCAGTCATTGTTAGCATTAATCGTGGACTTAGGTTTGTAA (SEQ ID NO: 14) Nasutitermes ATGAGATTTCCATCTATTTTCACTGCCGTCTTATTTGCAGCCTCAAGTGCT MRFPSIFTAVLFAASSALAAYDY walkeri EG TTAGCAGCCTATGATTACAAACAAGTATTGAGAGATTCCTTATTGTTCTAC KQVLRDLLFYEAQRSGRLPAD GAAGCTCAGAGAAGCGGTAGATTACCAGCAGACCAGAAGGTCACCTGGAG QKVTWRKDSALNDQGEQGQDL AAAAGATTCCGCCTTGAATGATCAGGGAGAGCAAGGTCAAGACTTAACCG TGGYFDAGDFVKFGFPMAYTAT GAGGTTATTTTGACGCCGGTGATTTTGTGAAGTTTGGATTCCCAATGGCTT VLAWGLIDFEAGYSSAGALDDG ATACAGCAACCGTTTTGGCCTGGGGTTTAATCGACTTTGAAGCCGGTTACT RKAVKWATDYFIKAHTSQNEFY

CTTCTGCTGGTGCCTTGGACGATGGTAGAAAAGCAGTAAAGTGGGCTACT GQVGQGDVDHAYWGRPEDMT GATTACTTTATAAAAGCCCATACTTCTCAAAACGAGTTTTACGGACAAGTC MARPAYKIDTSRPGSDLAGETA GGTCAGGGTGACGTAGATCACGCATATTGGGGACGTCCTGAAGATATGAC AALAAASIVFKNVDGTYSNNLL AATGGCTAGACCAGCCTACAAGATTGATACCAGCAGACCAGGTAGTGACT THARQLFDFANNYRGKYSDSIT TAGCAGGAGAAACTGCTGCAGCTTTGGCTGCCGCATCCATCGTTTTCAAGA DARNFYASADYRDELVWAAAW ATGTAGATGGTACATATTCCAACAACTTACTTACTCATGCTAGACAGTTGT LYRATNDNSYLNTAESLYNEFG TTGATTTCGCCAACAATTACAGAGGAAAATACTCTGATAGTATTACCGATG LQNWGGGLNWDSKVSGVQVLL CAAGAAACTTTTACGCTAGTGCCGACTATAGAGATGAGTTAGTCTGGGCA AKLTNKQEYKDTIQSYVNYLIN GCTGCCTGGTTGTACAGAGCAACCAACGACAATTCTTACTTGAACACTGCT NQQKTPKGLLYIDMWGTLRHA GAATCATTATACAACGAGTTTGGATTGCAAAATTGGGGTGGAGGGTTAAA ANAAFIMLEAADLGLSASSYRQ CTGGGATTCTAAAGTGAGTGGTGTTCAAGTTTTGTTAGCCAAGTTGACCAA FAQTQIDYALGDGGRSSFVCGFG CAAACAAGAGTATAAGGACACTATTCAATCATACGTGAATTACTTAATCA SNPPTRPHHRSSSCPPAPATCDW ATAACCAACAGAAAACTCCAAAGGGATTGTTATACATTGACATGTGGGGG NTFNSPDPNYNVLSGALVGGPD ACCTTGAGACACGCAGCTAACGCAGCCTTTATAATGTTAGAAGCTGCCGA QNDNYVDDRSDYVHNEVATDY CTTAGGTTTATCCGCTTCATCTTATAGACAGTTCGCCCAAACACAAATAGA NAGFQSALAALVALGY CTACGCATTGGGGGACGGTGGACGTTCTTTTGTCTGTGGTTTCGGTTCTAA (SEQ ID NO: 35) TCCTCCAACTAGACCTCATCATAGATCCAGTTCATGCCCGCCTGCTCCAGC TACCTGTGATTGGAATACATTCAATTCTCCTGACCCAAACTACAATGTTTT ATCCGGTGCCTTGGTTGGTGGTCCTGACCAGAATGATAACTACGTGGACG ATAGAAGTGATTATGTCCATAATGAGGTAGCAACTGACTACAATGCCGGT TTCCAATCAGCCTTAGCCGCTTTAGTCGCCTTAGGTTACTAA (SEQ ID NO: 15) Reticuli- ATGAGATTCCCAAGTATATTTACTGCCGTATTATTTGCAGCCTCCAGTGCA AB019095 termes TTAGCCGCTTATGACTACAAAACAGTATTGTCCAATTCCTTGTTGTTCTAC MRFPSIFTAVLFAASSALAAYDY speratus EG GAAGCTCAAAGATCCGGTAAGTTACCTTCTGACCAGAAAGTGACCTGGAG KTVLSNSLLFYEAQRSGKLPSDQ AAAGGATTCAGCATTAAACGACAAAGGACAAAAGGGTGAGGACTTAAC KVTWRKDSALNDKGQKGEDLT GGTGGATATTACGACGCCGGAGACTTTGTGAAATTTGGTTTTCAATGGCT GGYYDAGFVKFGFPMAYTVT TACACAGTTACCGTATTGGCATGGGGTGTTATTGATTACGAATCCGCCTAC VLAWGVIDYESSAYSAAGALDSG TCTCGCCGCAGGAGCTTTAGATTCAGGTAGAAAGGCCTTGAAATATGGGAC RKALKYGTDYFLKAHTAANEFY CGACTATTTCTTAAAGGCACATACAGCAGCTAACGAGTTTTACGGACAGG GQVGQGDVDHAYWGRPEDMT TGGGTCAAGGTGACGTTGACCACGCATACTGGGGACGTCCTGAAGATATG MSRPAYKIDTSKPGSDLAAETA ACCATGAGCAGACCAGCATACAAAATAGACACTTCTAAGCCTGGTTCCGA AALAATAIAYKSADATYSNNLIT CTTAGCTGCAGAGACTGCAGCTGCATTAGCAGCCACAGCTATTGCATACA HAKQLFDFANNYRGKYSDSITD AATCTGCCGATGCAACATATTCCAACAATTTGATAACACATGCAAAACAA AKNFYASGDYKDELVWAAAWL TTATTCGACTTTGCCAACAATTACAGAGGAAAATATTCCGATAGTATTACC YRATNDNTYLTKAESLYNEFGL GATGCCAAGAACTTTTATGCTTCTGGTGATTACAAAGACGAATTGGTATGG GNFNGAFNWDNKVSGVQVLLA GCCGCTGCATGGTTGTACAGAGCAACCAATGACAACACATATTTGACTAA KLTSKQVYKDKVQSYVDYLISS GGCAGAATCCTTATACAATGAATTTGGTTTGGGAAACTTCAATGGTGCCTT QKKTPKGLVYIDQWGTLRIIAA CAATTGGGATAACAAAGTCTCCGGAGTCCAGGTGTTATTGGCCAAGTTAA NSALIALQAADLGINAATYRAY CCTCAAAACAAGTGTATAAGGATAAGGTACAGTCTTACGTGGACTATTTG AKKQIDYALGDGGRSYVIGFGT ATCTCCTCACAAAAAAAGACACCAAAAGGTTTAGTGTACATCGATCAATG NPPVRPHHRSSSCPDAPAVCDW GGGTACTTTAAGACACGCAGCTAATTCTGCTTTGATCGCTTTGCAGGCAGC NTYNSAGPNAHVLTGALVGGPD TGACTTAGGAATTAACGCTGCTACTTACAGAGCCTACGCAAAGAAGCAAA SNDSYTDARSDYISNEVATDYN TCGACTATGCTTTGGGTGATGGTGGAAGATCCTATGTTATTGGATTTGGGA AGFQSAVAGLLKAGV CCAACCCTCCAGTAAGACCACATCACAGAAGTTCATCTTGCCCAGATGCA (SEQ ID NO: 36) CCAGCTGTCTGCGATTGGAACACCTATAACTCCGCTGGTCCAAACGCCCAC GTGTTAACCGGTGCATTGGTTGGAGGACCTGATAGTAATGATAGTTATACC GATGCTCGTTCTGACTACATATCCAACGAAGTGGCAACTGATTACAATGCG GGTTTCCAATCCGCTGTCGCTGGATTATTGAAGGCGGGTGTCTAA (SEQ ID NO: 16) Neosartorya ATGAGATTTCCATCTATTTTCACTGCAGTTTTGTTCGCAGCCAGTTCCGCT XM_001258277 fisheri EG TTGGCCCAACAGATCGGGTCCATCGCCGAAAATCATCCTGAGTTGACAACC MRFPSIFTAVLFAASSALAQQIG TATAGATGCTCCTCTCAAGCTGGATGCGTAGCACAGAGTACTTCCGTCGTG SIAENHPELTTYRCSSQAGCVAQ TTAGATATTAACGCTCATTGGATTCATCAAAACGGTGCCCAAACAAGTTGC STSVVLDINAHWIHQNGAQTSC ACTACCTCAAGTGGATTGGACCCTTCATTGTGCCCTGATAAAGTCACCTGT TTSSGLDPSLCPDKVTCSQNCVV TCTCAGAACTGCGTAGTCGAAGGAATAACCGACTACTCATCTTTTGGTGTG EGITDYSSFGVQNSGDAMTLRQ CAAAACTCCGGAGATGCAATGACATTAAGACAGTATCAAGTTCAAAATGG YQVQNGQIKTLRPRVYLLAEDG ACAGATCAAAACATTGCGTCCTAGAGTGTACTTGTTAGCTGAGGATGGAA INYSKLQLLNQEFTFDVDASKLP TCAATTACTCCAAATTGCAGTTGTTGAACCAAGAGTTTACTTTCGATGTGG CGMNGALYLSEMDASGGRSAL ACGCTTCCAAATTGCCTTGTGGTATGAATGGAGCTTTATATTTGTCAGAAA NPAGATYGTGYCDAQCFNPGP TGGATGCTTCTGGTGGACGTTCTGCCTTGAACCCAGCGGGTGCCACATATG WINGEANTAGAGACCQEMDLW GAACAGGTTACTGTGATGCCCAGTGCTTCAACCCAGGTCCATGGATAAAT EANSRSTIFSPHPCTTAGLYACT GGAGAAGCAAATACTGCTGGAGCCGGTGCATGTTGCCAAGAGATGGACTT GAECYSICDGYGCTYNPYELGA ATGGGAAGCCAACTCCCGTTCTACCATTTTCAGTCCTCACCCATGTACAAC KDYYGYGLIDTIDTAKPITVVTQF TGCGGGTTTGTATGCCTGTACTGGAGCTGAGTGCTACTCAATCTGTGACGG MTADNTATGTLAEIRRLYVQDG TTATGGTTGCACTTACAACCCTTATGAATTAGGAGCCAAAGATTACTATGG KVIGNTAVAMTEAFCSSSRTFEE TTACGGTTTGACTATTGACACCGCAAAGCCAATAACAGTGGTTACTCAGTT LGGLQRMGEALGRGMVPVFSI TATGACCGCTGATAATACAGCAACCGGTACATTAGCAGAGATCAGAAGAT WDDPGLWMHWLDSDGAGPCG TATATGTTCAAGATGGTAAAGTAATCGGAAATACAGCCGTGGCCATGACC NTEGDPAFIQANYPNTAVTFSKV GAGGCATTTTGTAGTTCTAGTAGAACATTTGAAGAGTTAGGTGGTTTGCAA RWGDIGSTYSS (SEQ ID NO: 37) AGAATGGGAGAAGCTTTAGGTAGAGGAATGGTGCCAGTTTTCTCAATATG GGACGATCCTGGTTTGTGGATGCATTGGTTAGATTCTGACGGTGCAGGACC TTGTGGTAATACTGAAGGTGATCCTGCCTTCATTCAGGCTAACTACCCAAA TACCGCCGTAACATTCTCCAAGGTGAGATGGGGAGATATCGGTTCTACCTA TAGTTCTTAA (SEQ ID NO: 17) Reticuli- ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCT DQ014512 termes TTAGCACAATGGATGCAGATCGGTGGTAAGCAGAAATATCCTGCCTTTAAG MRFPSIFTAVLFAASSALAQWM flavipes EG CCAGGTGCTAAGTACGGAAGAGGTTATTGTGACGGACAGTGCCCTCACGA QIGGKQKTPAFKPGAKYGRGYC CATGAAGGTGTCTAGTGGAAGAGCAAACGTTGACGGATGGAAGCCACAA DGQCPHDMKVSSGRANVDGWK GACAACGACGAAAATAGTGGAAATGGAAAATTGGGTACATGTTGCTGGGA PQDNDENSGNGKLGTCCWEMD GATGGATATATGGGAAGGAAACTTAGTGTCCCAAGCCTACACCGTTCACG IWEGNLVSQAYTVHAGSKSGQY CTGGTTCCAAGTCCGGACAATATGAGTGTACTGGAACACAATGCGGTGAC ECTGTQCGDTDSGERFKGTCDK ACCGACAGTGGTGAAAGATTCAAGGGAACATGCGATAAAGATGGTTGTGA DGCDFASYRWGATDYYGPGKT TTTCGCAAGTTACAGATGGGGAGCTACAGACTATTACGGTCCTGGAAAGA VDTKQPMTVVTQFIGDPLTEIKR CCGTGGACACCAAACAGCCAATGACAGTCGTGACCCAGTTCATTGGTGAC VYVQGGKVINNSKTSNLGSVYD CCTTTGACTGAGATAAAGAGAGTTTATGTACAAGGAGGAAAAGTCATAAA SLTEAFCDDTKQVTGDTNDFKA CAATTCCAAAACATCTAACTTAGGTTCAGTGTACGATTCTTTGACTGAGGC KGGMSGFSKNLDTPQVLVMSL CTTCTGCGATGACACCAAACAGGTTACAGGTGATACAAATGACTTTAAGG WDDHTANMLWLDSTYPTDSTK CTAAAGGAGGTATGTCTGGATTCTCCAAGAACTTAGACACCCCACAAGTTT PGAARGTCAVTSGDPKDVESKQ TGGTGATGTCTTATGGGATGACCATACAGCTAATATGTTATGGTTAGATT ANSQVVYSDIKFGPINSTYKAN CTACTTATCCTACCGATAGTACAAAGCCAGGTGCCGCAAGAGGTACTTGT (SEQ ID NO: 38) GCCGTCACCTCCGGGGACCCTAAAGATGTGGAATCCAAGCAAGCCAACTC TCAGGTAGTTTACAGTGACATTAAGTTTGGTCCTATTAATTCAACATACAA AGCAAATTAA (SEQ ID NO: 18) Trichoderma ATGGTCTCCTTCACCTCCCTGCTGGCCGGCGTTGCCGCTATCTCTGGTGTC AB003694 reesie EG1 CTAGCAGCCCCTGCCGCAGAAGTTGAACCTGTCGCAGTTGAGAAACGTGAG MVSFTSLLAGVAAISGVLAAPA GCCGAAGCAGAAGCTCAACAACCAGGAACATCAACACCAGAAGTCCATC AEVEPVAVEKREAEAEAQQPGT CAAAGTTAACAACCTATAAATGTACTAAGAGTGGAGGGTGTGTAGCGCAG STPEVHPKLTTYKCTKSGGCVA GACACAAGTGTGGTCTTAGACTGGAATTATCGTTGGATGCATGATGCCAAT QDTSVVLDWNYRWMHDANYN TATAATTCCTGTACTGTTAACGGCGGTGTTAACACTACGTTATGCCCCGAT SCTVNGGVNTTLCPDEATCGKN GAAGCGACTTGTGGTAAGAATTGTTTTATTGAAGGGGTTGACTACGCCGCT CFIEGVDYAASGVTTSGSSLTMN AGTGGTGTTACGACGAGTGGGTCATCCTTGACGATGAATCAATACATGCCT QYMPSSSGGYSSVSPRLYLLDSD TCTTCTAGTGGTGGGTATTCCTCTGTGTCTCCAAGGCTGTATTATTGGATT GEYVMLKLNGQELSFDVDLSAL CCGATGGGGAATATGTTAAAATTAAATGGGCAAGAACTGAGTTTT PCGENGSLYLSQMDENGGANQ GATGTGGATCTATCTGCATTACCTTGTGGAGAAAATGGTAGTCTTTATTTA YNTAGANYGSGYCDAQCPVQT TCACAAATGGACGAAAACGGCGGAGCCAATCAGTACAATACAGCTGGTGC WRNGTLNTSHQGFCCNEMDILE TAATTATGGTTCAGGCTATTGTGATGCTCAATGTCCAGTGCAGACTTGGAG GNSRANALTPHSCTATACDSAG GAATGGCACCTTAAACACATCACATCAAGGATTTTGCTGTAACGAAATGG CGFNPYGSGYKSYYGPGDTVDT ACATATTAGAAGGTAATTCAAGAGCTAATGCACTAACTCCGCACTCTTGTA SKTFTIITQFNTDNGSPSGNLVSI CTGCGACCGCATGTGATTCTGCCGGTTGTGGTTTCAACCCTTATGGTTCTG TRKYQQNGVDIPSAQPGGDTISS GTTATAAGAGTTACTACGGTCCGGGAGACACCGTGGATACGTCAAAGACC CPSASAYGGLATMGKALSSGM TTCACTATAATCACTCAGTTTAACACGATAACGGATCTCCGAGTGGTAAT VLVFSIWNDNSQYMNWLDSGN TTGGTGAGTATTACTAGGAAATATCAGCAGAACGTGTTGATATTCCGTCC AGPCSSTEGNPSNILANNPNTHV GCGCAGCCAGGCGGTGACACTATATCTAGCTGTCCTTCCGCCAGTGCCTAT VFSNIRWGDIGSTTNSTAPPPPPA GGCGGACTTGCTACAATGGGTAAGGCATTGTCCTCAGGTATGGTCCTAGTA SSTTFSTTRRSSTTSSSPSCTQTH TTTTCTATTTGGAATGATAATTCACAATACATGAATTGGCTGGATTCTGGT WGOCGGIGYSGCKTCTSGTTCQ AATGCAGGCCCTTGCTCCTCTACAGAAGGTAACCCAAGCAATATACTAGC YSNDYYSQC (SEQ ID NO: 39) TAATAACCCAAATACTCATGTTGTCTTTAGTAATATTAGATGGGGCGATAT AGGTAGCACTACGAACAGTACCGCACCTCCTCCTCCACCTGCTAGCTCCAC GACATTTTCCACTACTAGAAGGTCCAGCACTACCAGCTCATCACCATCTTG TACTCAAACCCATTGGGGACAGTGTGGTGGTATAGGTTACAGCGGTTGCAA AACTTGCACATCTGGTACTACATGCCAATACAGTAATGACTATTAACTCAC AATGTTAA (SEQ ID NO: 19) Aspergillus TGGTTTCTGCTTTGCCATCTAGACAAATGAAAAAGAGGGATTCTGGTTTTA MRISNLIVAASAATMVSALPSRQ kawachii AATGGGTTGGTACTTCTGAATCTGGTGCTGAATTTGGTTCTGCTTTACCAG MKKRDSFKWVGTSESGAEFGS EgA GTACTTTGGGTACTGATTATACTTGGCCAGAAACTTCTAAAATTCAAGTTT ALPGTLGTDYTWPETSKIQVLR TGAGAAACAAGGGTATGAACATTTTTAGAATACCATTCTTGATGGAAAGAT NKGMNIFRIPFLMERLTPDGLTG TAACTCCAGATGGTTTGACTGGTTCTTTTGCTTCTACTTACTTGTCTGATT SFASTYLSDLKSTVEFVTNSGAY TGAAGTCAACTGTTGAATTTGTTACTAATTCTGGTGCTTATGCTGTTTTAG AVLDPHNYGRFDGIIESTSDFK ATCCACATAATTACGGTAGATTCGTGGTTCTATTATTGAATCTACTTCTGA TWWKNVATEFADNDKVIFDTN TTTTAAGACTTGGTGGAAAAATGTTGCTACTGAATTTGCTGATAACGATAA NEYHDMEQSLVLNLNQAAINGI GGTTATTTTCGATACAAACAACGAATATCATGATATGGAACAATCTTTGGT RAAGATTQYIFVEGNAYTGAW TTTGAATTGAACCAAGCTGCTATTAATGGTATTAGAGCTGCTGGTGCTACT DWTTYNDDLSGLTDSEDKIIYE ACTCAATACATTTTCGTTGAAGGTAATGCTTATACTGGTGCTTGGGATTGG MHQYLDSDSSGTSETCVSSTIGK ATATACGAAATGCATCAATACTTGGATTCTGATTCTTCTGGTACATCTGAA ERIEKATEWLKTNNKQGIIGEFA ACTTGTGTTTCTTCTACTATTGGTAAAGAAAGAATTGAAAAGGCTACTGAA GGVNSVCEEAVEGMLAYMSEN TGGTTGAAAACTAACAACAAGCAAGGTATTATTGGTGAATTTGCAGGTGGT SDVWVGASWWSAGPWWGTYM GTTAATTCTGTTTGTGAAGAGGCTGTTGAAGGAATGTTGGCTTTATATGTC YSLEPTDGTAYSTYLPILEKYFPS TGAAAATTCTGATGTTTGGGTTGGTGCTTCTTGGTGGTCTGCTGGTCCATG GDASSSSSASASVAAATSAVSTT GTGGGTACTTACATGTATTCTTTGGAACCAACTGATGGTACTGCTTATTCT TTSSFEQTTTPATQVEIASSSSSS ACTTATTTGCCAATTTTGGAAAATACTTCCCATCTGGTGATGCTTCATCAT SAVAASQTTLSKVKSKSKSPVKL CTTCATCTGCTTCAGCTTCAGTTGCAGCCGCTACTTCTGCTGTTTCTACTA SSATSSAVSSAAAVTTPAVAATT CTACTACAGCTGCATTTGAACAAACTACTACTCCAGCTACTCAAGTTGAAA PAAAPTSSVAFATTSVYVPTTT TTGCTTCTTCTTCATCTTCATCATCAGCTGTGCTGCTTCACAAACTACTTT AAAPSQVSSSAAASSSGVVGVS GTCTAAGGTTAAGTCTAAACTAAATCTCCATGTAAATTGTCATCGCTACTT DPQGPSATNSAGEVNQYYQCGG CATCTGCTGTTTCATCAGCTGCTGCAGTTACTACACCTGCAGTTGCAGCTA INWTGPTVCASPYTCKVQNDYY CAACTCCAGCTGCTGCTCCAACTTCTTCTTCTGTTGCTTTTGCTACTACTT YQCVAE (SEQ ID NO: 52) CTGTTTACGTTCCAACTACTACTGCTGCTGCACCATCTCAAGTTTCATCTT CAGCTGCAGCTTCATCTTCAGGTGTTGTTGGTGTTTCTGATCCACAAGGTC

CATCTGCTACTAATTCTGCTGGTGAAGTTAATCAATATTACCAATGTGGTG GTATTAATTGGACTGGTCCAACTGTTTGTGCTTCTCCATATACTTGTAAGG TTCAAAACGATTACTACTATCAATGTGTTGCTGAATTATAAGGCGCGCC (SEQ ID NO: 47) Heterodera TTAATTAAAATGCATTGGGCTGATGTTGCTTGTTCTAGACCACCATGGCCA MHWADVACSRPPWPRDSVKAL schachtii AGAGATTCTGTTAAAGCTTTGAAGTGTAATTGGAACGCTAATGTTATTAGA KCNWNANVIRGAMGVDEGGYL Eng1 GGTGCTATGGGTGTTGATGAAGGTGGTTATTTGTCTGATGCTAATACTGCT SDANTAYNLMVAVIEAAISNGIY TACAATTTGATGGTTGCTGTTATTGAAGCTGCTATTTCTAATGGTATCTACG VIVDWHAHNAHPDEAVKFFTRI TTATTGTTGATTGGCATGCTCATAATGCTCATCCAGATGAAGCTGTTAAAT AQAYGSYLHILYEDFNEPLDVS TCTTTACTAGAATTGCTCAAGCTTATGGTTCTTACTTGCATATTTGTACGA WTDVLVPYHKKVIAAIRAIDKK AGATTTCAATGAACCATTGGATGTTTCTTGGACTGATGTTTTGGTTCCATA NVIILGTPKWSQDVDVASQNPIK CCATAAAAAAGTTATTGCTGCCATTAGAGCTATTGATAAGAAGAACGTTA DYQNLMYTLHFYASSHFTSDLG TTATCTTGGGTACTCCAAAATGGTCACAAGATGTTGATGTTGCTTCTCAAA AKLKTAVNNGLPVFVTEYGTCE ATCCAATTAAGGATTACCAAAACTTGATGTACACTTTGCATTTTTACGCTT ASGNGNLNTDSMSSWWTLLDS CATCTCATTTTACATCTGATTTGGGTGCTAAATTGAAAACTGCTGTTAACA LKISYANWAISDKSEACSALSPG ATGGTTTGCCAGTTTTTGTTACTGAATATGGTACTTGTGAAGCTTCTGGTA TTAVNVGVSSRWTSSGNMVAS ATGGTAATTTGAATACTGATTCTATGTCATCTTGGTGGACTTTGTTGGATTC YYKKKSTGISCSGSSSGSSSGSSS TTTGAAAATTTCTTACGCTAATTGGGCTATTTCTGATAAATCTGAAGCTTGT GSSGTSSGSSGSSSGSSSGSSSGS TCTGCTTTGTCTCCAGGTACTACTGTTAATGTTGGTGTTTCTTCTAGAT SGSSSGSSSGSGSASISVVPSNTW GGACTTCTTCTGGTAATATGGTTGCTTCTTACTACAAAAAAAAGTCCACTG NGGGRVNFEIKNTGSVPLCGVV GTATTTCTTGTTCTGGTAGTTCTTCAGGTTCTTCAAGTGGTTCATCTAGTGG FSVSLPSGTTLGGSWNMESAGS TTCTTCCGGTACATCTTCTGGTTCTAGTGGTTCATCTAGTGGTAGTTCTTCC GQYSLPSWVRIEAGKSSKDAGL GGTAGTTCTAGTGGTAGTTCTGGTTCAAGTTCTGGTTCCTCCTCTGGTTCTG TFNGKDKPTAKIVTTKKC GTTCTGCATCTATTTCTGTTGTTCCATCTAATACTTGGAATGGTGGTGGTAG (SEQ ID NO: 53) AGTTAATTTTGAAATTAAGAACACTGGTTCTGTTCCATTGTGTGGTGTTGTT TTTTCTGTTTCTTTGCCATCTGGTACTACTTTGGGTGGTTCTTGGAATATGG AATCTGCTGGTTCTGGTCAATATTCTTTACCATCTTGGGTTAGAATTGAAG CTGGTAAATCTTCTAAAGATGCTGGTTTGACTTTTAATGGTAAAGATAAGC CAACTGCTAAAATTGTTACCACCAAGAAGTGCTTATAAGGCGCGCC (SEQ ID NO: 48) Hypocrea TTAATTAAAATGAACAAGTCTGTTGCTCCATTGTTGTTGGCTGCTTCTATTT MNKSVAPLLLAASILYGGAVAQ jecorina TGTATGGTGGTGCTGTTGCTCAACAAACTGTTTGGGGTCAATGTGGTGGTA QTVWGQCGGIGWSGPTNCAPGS (anamorph: TTGGTTGGTCTGGTCCAACTAATTGTGCTCCAGGTTCTGCTTGTTCTACTTT ACSTLNPYYAQCIPGATTITTSTR Trichoderma GAATCCATATTATGCTCAATGTATTCCAGGTGCTACTACTATTACTACTTCT PPSGPTTTTRATSTSSSTPPTSSG reesei) Eg2 ACTAGACCACCATCTGGTCCAACAACTACTACTAGAGCTACTTCTACATCT VRFAGVNIAGFDFGCTTDGTCV TCTTCTACTCCACCAACTTCATCTGGTGTTAGATTTGCTGGTGTTAACATTG TSKVYPPLKNFTGSNNYPDGIGQ CTGGTTTTGATTTTGGTTGTACTACTGATGGTACTTGTGTTACTTCTAAAGT MQHFVNEDGMTIFRLPVGWQY TTACCCACCATTGAAAAATTTCACTGGTTCTAACAATTATCCAGATGGTAT LVNNNLGGNLDSTSISKYDQLV TGGTCAAATGCAACATTTTGTTAACGAAGATGGTATGACTATTTTTAGATT QGCLSLGAYCIVDIHNYARWNG GCCAGTTGGTTGGCAATATTTGGTTAACAACAATTTGGGTGGTAATTTGGA GIIGQGGPTNAQFTSLWSQLASK TTCTACTTCTATTTCTAAGTACGATCAATTGGTTCAAGGTTGTTTGTCTTTG YASQSRVWFGIMNEPHDVNINT GGTGCTTACTGTATTGTTGATATTCATAATTATGCTAGATGGAATGGTGGT WAATVQEVVTAIRNAGATSQFI ATTATTGGTCAAGGTGGTCCAACAAATGCTCAATTTACTTCTTTGTGGTCA SLPGNDWQSAGAFISDGSAAAL CAATTGGCTTCAAAATATGCTTCTCAATCTAGAGTTTGGTTTGGTATTATG SQVTNPDGSTTNLIFDVHKYLDS AATGAACCACATGATGTTAACATTAATACTTGGGCTGCTACTGTTCAAGAA DNSGTHAECTTNNIDGAFSPLAT GTTGTTACTGCTATTAGAAATGCTGGTGCTACTTCTCAATTCATTTCTTTGC WLRQNNRQILTETGGGNVQSC CAGGTAATGATTGGCAATCTGCTGGTGCTTTTATTTCTGATGGTTCTGCTGC IQDMCQQIQYLNQNSDVYLGYV TGCTTTGTCTCAAGTTACTAATCCAGATGGTTCTACTACTAATTTGATCTTC GWGAGSFDSTYVLTETPTSSGN GATGTTCATAAGTACTTGGATTCTGATAATTCTGGTACTCATGCTGAATGT SWTDTSLVSSCLARK ACTACAAACAATATTGATGGTGCTTTTTCTCCATTGGCTACTTGGTTGAGA (SEQ ID NO: 54) CAAAACAATAGACAAGCTATTTTGACTGAAACTGGTGGTGGTAATGTTCA ATCTTGTATCCAAGATATGTGCCAACAAATTCAATACTTGAACCAAAATTC TGATGTTTATTTGGGTTACGTTGGTTGGGGTGCTGGTTCTTTTGATTCTACT TACGTTTTAACTGAAACTCCAACTTCTTCTGGTAATTCTTGGACTGATACTT CTTTGGTTTCTTCATGTTTGGCTAGAAAGTTATAAGGCGCGCC (SEQ ID NO: 49) Orpinomyces TTAATTAAAATGAAGTTCTTGAACTCTTTGTCTTTGTTGGGTTTGGTTATTG MKFLNSLSLLGLVIAGCEAMRNI sp. PC-2 CelB CTGGTTGTGAAGCTATGAGAAACATTTCTTCTAAAGAATTGGTTAAAGAAT SSKELVKELTIGWSLGNTLDASC TGACTATTGGTTGGTCTTTGGGTAATACTTTGGATGCTTCTTGTGTTGAAAC VETLNYSKDQTASETCWGNVKT TTTGAACTACTCTAAAGATCAAACTGCTTCTGAAACTTGTTGGGGTAATGT TQELYYKLSDLGFNTFRIPTTWS TAAAACTACTCAAGAATTGTACTACAAATTGTCTGATTTGGGTTTCAATAC GHFGDAPDYKISDVWMKRVHE TTTCAGAATACCAACTACTTGGTCTGGTCATTTTGGTGATGCTCCAGATTA VVDYALNTGGYAILNIHHETWN CAAAATTTCTGATGTTTGGATGAAAAGAGTTCACGAAGTTGTTGATTATGC YAFQKNLESAKKILVAIWKQIA TTTGAATACTGGTGGTTACGCTATTTTGAACATTCATCATGAAACTTGGAA AEFGDYDEHLIFEGMNEPRKVG TTACGCTTTTCAAAAGAATTTGGAATCTGCTAAAAAGATTTTGGTTGCTAT DPAEWTGGDQEGWNFVNEMN TTGGAAACAAATTGCTGCTGAATTTGGTGATTACGATGAACATTTGATTTT ALFVKTIRATGGNNANRHLMIP TGAAGGTATGAATGAACCAAGAAAAGTTGGTGATCCAGCTGAATGGACTG TYASSVNDGSINNFKYPNGDDK GTGGTGATCAAGAAGGTTGGAATTTTGTTAATGAAATGAACGCTTTGTTCG VIVSLHSYSPYNFALNNGPGAIS TTAAAACTATTAGAGCTACTGGTGGTAACAATGCTAATAGACATTTGATGA NFYDGNEIDWVMNTINSSFISKG TTCCAACTTATGCTGCTTCTGTTAATGATGGTTCTATTAACAATTTTAAGTA IPVIIGEFVAMNRDNEDDRERW CCCAAATGGTGATGATAAAGTTATTGTTTCTTTGCATTCTTACTCTCCATAC QEYYIKKATALGIPCVIWDNGYF AATTTTGCTTTGAACAATGGTCCAGGTGCTATTTCTAATTTCTACGATGGT EGEGERFGIIDRKSLNVIFPKLIN AACGAAATTGATTGGGTTATGAACACTATTAACTCTTCATTCATTTCTAAG GLMKGLGDEKPKTTIRRTTTTT GGTATTCCAGTTATTATTGGTGAATTTGTTGCTATGAACAGAGATAATGAA VQVQPTINNECFSTRLGYSCCNG GATGATAGAGAAAGATGGCAAGAATACTACATTAAAAAGGCTACTGCTTT FDVLYTDNDGQWGVENGNWC GGGTATTCCATGTGTTATTTGGGATAATGGTTATTTTGAAGGTGAAGGTGA GIKSSCGNNQRQCWSERLGYPC AAGATTTGGTATTATTGATAGAAAGTCTTTGAACGTTATTTTCCCAAAGTT CQYTTNAEYTDNDGRWGVENG GATTAATGGTTTGATGAAAGGTTTGGGTGATGAAAAACCAAAAACTACTA NWCGIY (SEQ ID NO: 55) TTAGAAGAACTACTACTACTACAGTTCAAGTTCAACCAACTATTAACAACG AATGTTTCTCTACTAGATTGGGTTATTCTTGTTGTAATGGTTTCGATGTTTT GTACACTGATAATGATGGTCAATGGGGTGTTGAAAATGGTAATTGGTGTG GTATTAAATCTTCTTGTGGTAACAATCAAAGACAATGTTGGTCTGAAAGAT TAGGTTATCCATGTTGTCAATACACTACTAATGCTGAATATACAGACAACG ACGGTAGATGGGGTGTAGAAAACGGTAACTGGTGCGGAATATACTTGTAA GGCGCGCC (SEQ ID NO: 50) Irpex lacteus TTAATTAAAATGAAGTCTTTGTTGTTGTCTGCTGCTGCTACTTTGGCTTTAT MKSLLLSAAATLALSTPAFSVSV En1 CTACTCCAGCTTTTTCTGTTTCTGTTTGGGGTCAATGTGGTGGTATTGGTTT WGQCGGIGFTGSTTCDAGTSCV TACTGGTTCTACTACTTGTGATGCTGGTACTTCTTGTGTTCATTTGAACGAT HLNDYYFQCQPGAATSTVQPTT TACTACTTTCAATGTCAACCAGGTGCTGCTACTTCTACTGTTCAACCAACT TASSTSSAAAPSSSGNAVCSGTR ACTACTGCTTCTTCTACTTCTTCTGCTGCAGCTCCATCTTCTTCAGGTAATG NKFKFFGVNESGAEFGNNVIPGT CTGTTTGTTCTGGTACTAGAAACAAGTTTAAGTTCTTCGGTGTTAATGAAT LGTDYTWPSPSSIDFFVGKGFNT CTGGTGCTGAATTTGGTAACAATGTTATTCCAGGTACTTTGGGTACTGATT FRVPFLMERLSPPATGLTGPFDS ATACTTGGCCATCTCCATCTTCTATTGATTTTTTCGTTGGTAAGGGTTTTAA TYLQGLKTIVSYITGKGGYALV TACTTTCAGAGTTCCATTTTTGATGGAAAGATTGTCTCCACCTGCTACTGGT DPHNFMIYNGATISDTNAFQTW TTGACTGGTCCATTTGATTCTACTTATTTGCAAGGTTTGAAAACTATTGTTT WQNLAAQFKTDSHVVFDVMNE CTTACATTACTGGTAAAGGTGGTTATGCTTTGGTTGATCCACATAACTTA PHDIPAQTVFNLNQAAINRIRAS TGATTTACAACGGTGCTACTATTCTGATACTAATGCTTTTCAAACTTGGTG GATSQSILVEGTSYTGAWTWTT GCAAAATTTGGCTGCTCAATTTAAGACTGATTCTCATGTTGTTTTCGATGTT TSGNSQVFGAIHDPNNNVAIEM ATGAATGAACCACATGATATTCCAGCTCAAACTGTTTTTAACTTGAACCAA HQYLDSDGSGTSPTCVSTIGAE GCTGCTATTAATAGAATTAGAGCTTCTGGTGCTACTTCTCAATCTATTTTGG RLQAATQWLQQNNLKGFLGEIG TTGAAGGTACTTCTTATACTGGTGCTTGGACTTGGACTACTACTTCTGGTA AGSNADCISAVQGALCEMQQSD ATTCTCAAGTTTTTGGTGCTATTCATGATCCAAACAACAATGTTGCTATTG VWLGALWWAAGPWWGDYFQS AAATGCATCAATACTTGGATTCTGATGGTTCTGGTACTTCTCCAACTTGTG IEPPSGVAVSSILPQALEPFL TTTCTCCAACTATTGGTGCTGAAAGATTGCAAGCTGCTACTCAATGGTGC (SEQ ID NO: 56) AACAAAACAATTTGAAAGGTTTCTTGGGTGAAATTGGTGCTGGTTCTAATG CTGATTGTATTTCTGCTGTTCAAGGTGCTTTGTGTGAAATGCAACAATCTG ATGTTTGGTTGGGTGCTTTGTGGTGGGCTGCTGGTCCATGGTGGGGTGATT ATTTTCAATCTATTGAACCACCATCTGGTGTTGCTGTTTCTTCTATTTTGCC ACAAGCTTTGGAACCATTTTTGTTATAAGGCGCGCC (SEQ ID NO: 51) .beta.-Glucosidases S. f. BGLI ATGGTCTCCTTCACCTCCCTCCTCGCCGGCGTCGCCGCCATCTCGGGCGTC FJ028723 TTGGCCGCTCCCGCCGCCGAGGTCGAATCCGTGGCTGTGGAGAAGCGCTC MVSFTSLLAGVAAISGVLAAPA GGACTCGCGAGTCCCAATTCAAAACTATACCCAGTCTCCATCCCAGAGAG AEVESVAVEKRSDSRVPIQNYT ATGAGAGCTCCCAATGGGTGAGCCCGCATTATTATCCAACTCCACAAGGT QSPSQRDESSQWVSPHYYPTPQ GGTAGGCTCCAAGACGTCTGGCAAGAAGCATATGCTAGAGCAAAAGCCAT GGRLQDVWQEAYARAKAIVGQ CGTTGGCCAGATGACTATTGTTGAAAAGGTCAATTTGACCACTGGTACCGG MTIVEKVNLTTGTGWQLDPCVG TTGGCAATTAGATCCATGTGTTGGTAATACCGGTTCTGTTCCAAGATTCGG NTGSVPRFGIPNLCLQDGPLGVR CATCCCAAACCTTTGCCTACAAGATGGGCCATTGGGTGTTCGATTCGCTGA FADFVTGYPSGLATGATFNKDL CTTTGTTACTGGCTATCCATCCGGTCTTGCTACTGGTGCAACGTTCAATAA FLQRGQALGHEFNSKGVHIALG GGATTTGTTTCTTCAAAGAGCTCAAGCTCTCGGTCATGAGTTCAACAGCAA PAVGPLGVKARGGRNFEAFGSD AGGTGTACATATTGCGTTGGGCCCTGCTGTTGGCCCACTTGGTGTCAAAGC PYLQGTAAAATIKGLQENNVMA CAGAGGTGGCAGAAATTTCGAAGCCTTTGGTTCCGACCCATATCTCCAAG CVKHFIGNEQEKYRQPDDINPAT GTACTGCTGCTGCTGCAACCATCAAAGGTCTCCAAGAGAATAATGTTATG NQTTKEAISANIPDRAMHELYL AGATGACATAAACCCTGCCACCAACCAAACTACTAAAGAAGCTATTAGTG WPFADSVRAGVGSVMCSYNRV CCAACATTCCAGACAGAGCCATGCATGAGTTGTACTTGTGGCCATTTGCCG NNTYACENSYMMNHLLKEELG ATTCGGTTCGAGCAGGTGTTGGTTCTGTTATGTGCTCTTATAACAGAGTCA FQGFVVSDWGAQLSGVYSAISG ACAACACTTACGCTTGCGAAAACTCTTACATGATGAACCACTTGCTTAAAG LDMSMPGEVYGGWNTGTSFWG AAGAGTTGGGTTTTCAAGGCTTTGTTTGTTTCGGACTGGGGTGCACAATTAA QNLTKAIYNETVPIERLDDMATR GTGGGGTTTATAGCGCTATCTCGGGCTTAGATATGTCTATGCCTGGTGAAG ILAALYATNSFPTEDHLPNFSSW TGTATGGGGGATGGAACACCGGCACGTCTTTCTGGGGTCAAAACTTGACG TTKEYGNKYYADNTTEIVKVNY AAAGCTATTTACAATGAGAACTGTTCCGATTGAAAGATTAGATGATATGGC HVDPSNDFTEDTALKVAEESIVL AACCAGGATCTTGGCTGCTTTGTATGCTACCAATAGTTTCCCAACAGAAGA LKNENNTLPISPEKAKRLLLSGIA TCACCTTCCAAATTTTTCTTCATGGACAACGAAAGAATAGGCAATAAATA AGPDPIGYQCEDQSCTNGALFQ TTATGCTGACAACACTACCGAGATTGTCAAAGTCAACTACCATGTGGACCC GWGSGSVGSPKYQVTPFEEISYL ATCAAATGACTTTACGGAGGACACAGCTTTGAAGGTTGCTGAGGAATCTA ARKNKMQFDYIRESYDLAQVTK TTGTGCTTTTAAAAAATGAAAACAACACTTTGCCAATTTCTCCCGAAAAGG VASDAHLSIVVVSAASGEGYITV CTAAAAGATTACTATTGTCGGGTATTGCTGCAGGCCCTGATCCGATAGGTT DGNQGDRRNLTLWNNGDKLIET ATCAGTGTGAAGATCAATCTTGCACAAATGGCGATTTGTTTCAAGGTTGGG VAENCANTVVVVTSTGQINFEG GTTCTGGCAGTGTTGGTTCTCCAAAATATCAAGTCACTCCATTTGAGGAAA FADHPNVTAIVWAGPLGDRSGT TTTCTTATCTTGCAAGAAAAAACAAGATGCAATTTGATTATATTCGGGAGT AIANILFGKANPSGHLPFTIAKTD CTTACGACTTAGCTCAAGTTACTAAAGTAGCTTCCGATGCTCATTTGTCTA DDYIPIETYSPSSGEPEDNHLVEN TAGTTGTTGTCTCTGCTGCAAGCGGTGAGGGTTATATAACCGTTGACGGTA DLLVDYRYFEEKNIWPRYAFGY ACCAAGGTGACAGAAGAAATCTCACTTTGTGGAACAACGGTGATAAATTG GLSYNEYEVSAKVSAAKKVDE ATTGAAACAGTTGCTGAAAACTGTGCCAATACTGTTGTTGTTGTTACTTCT

ELPEPATYLSEFSYQNAKDSKNP ACTGGTCAAATTAATTTTGAAGGCTTTGCTGATCACCCAAATGTTACCGCA SDAFAPTDLNRVNEYLYPYLDS ATTGTCTGGGCCGGCCCATTAGGTGACAGATCCGGGACTGCTATCGCCAAT NVTLKDGNYEYPDGYSTEQRTT ATTCTTTTTGGTAAAGCGAACCCATCAGGTCATCTTCCATTCACTATTGCTA PIQPGGGLGGNDALWEVAYKVE AGACTGACGATGATTACATTCCAATTGAAACCTACAGTCCATCGAGTGGT VDVQNLGNSTDKFVPQLYLKHP GAACCTGAAGACAACCACTTGGTTGAAAATGACTTGCTTGTTGACTATAG EDGKFETPIQLRGFEKVELSPGE ATATTTTGAAGAGAAGAATATTGAGCCAAGATACGCATTTGGTTATGGCTT KKTVFFELLRRDLSVWDTTRQS GTCTTACAATGAGTATGAAGTTAGCAATGCAAAGGTCTCGGCAGCCAAAA WIVESGTYEALIGVAVNDIKTSV AAGTTGATGAGGAGTTGCCCTGAACCAGCTACCTACTTATCGGAGTTTAGCT LFTI (SEQ ID NO: 40) ATCAAAATGCAAAAGACAGCAAAAATCCAAGTGATGCTTTTGCTCCAACA GATTTAAACAGAGTTAATGAGTACCTTTATCCATATTTAGATAGCAATCTT ATCAAAATGCAAAAGACAGCAAAAATCCAAGTGATGCTTTTGCTCCAACA GATTTAAACAGAGTTAATGAGTACCTTTATCCATATTTAGACAATGTT ACCTTAAAAGACGGAAACTATGAGTATCCCGATGGCTACAGCACTGAGCA AAGAACAACACCTATCCAACCTGGGGGCGGCTTGGGAGGCAACGATGCTT TGTGGGAGGTCGCTTATAAAGTTGAAGTGGACGTTCAAAAGTTGGGTAAC TCCACTGATAAGTTTGTTCCACAGTTGTATTTGAAACACCCTGAGGATGGC AAGTTTGAAACCCCTATTCAATTGAGAGGGGTTTGAAAAGGTTGAGTTGTCC CCGGGTGAGAAGAAGACAGTTGAGTTTGAGCTTTTGAGAAGAGATCTTAG TGTGTGGGATACCACCAGACAGTCTTGGATCGTTGAATCTGGTACTTATGA GGCCTTAATTGGTGTTGCTGTTAATGATATCAAGACATCTGTCCTGTTTACT ATT (SEQ ID NO: 20)

[0137] In certain aspects of the invention, the polypeptides and polynucleotides of the present invention are provided in an isolated form, e.g., purified to homogeneity.

[0138] The present invention also encompasses polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% similar to the polypeptide of any of SEQ ID NOs: 21-40, 46, or 52-56 and to portions of such polypeptide with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

[0139] As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.

[0140] The present invention further relates to a domain, fragment, variant, derivative, or analog of the polypeptide of any of SEQ ID NOs: 21-40, 46, or 52-56.

[0141] Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis. Therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.

[0142] Fragments of cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides encompass domains, proteolytic fragments, deletion fragments and in particular, fragments of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinonmyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides which retain any specific biological activity of the cellobiohydrolase, endoglucanase or beta-glucosidase proteins. Polypeptide fragments further include any portion of the polypeptide which retains a catalytic activity of cellobiohydrolase, endoglucanase or beta-glucosidase proteins.

[0143] The variant, derivative or analog of the polypeptide of any of SEQ ID NOs: 21-40, 46, or 52-56, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e., not membrane bound, yet still binds ligands to the membrane bound receptor. Such variants, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0144] The polypeptides of the present invention further include variants of the polypeptides. A "variant" of the polypeptide can be a conservative variant, or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein, A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.

[0145] By an "allelic variant" is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtti, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase protein.

[0146] The allelic variants, the conservative substitution variants, and members of the endoglucanase, cellobiohydrolase or .beta.-glucosidase protein families, can have an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95% amino acid sequence identity with a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinonmyces sp., Irpex lacteus, Acremonium thermophilum, R. flavipes, or Neosartorya fischeri cellobiohydrolase, endoglucanase or beta-glucosidase amino acid sequence set forth in any one of SEQ ID NOs: 21-40, 46, or 52-56. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

[0147] Thus, the proteins and peptides of the present invention include molecules comprising the amino acid sequence of SEQ ID NOs: 21-40, 46 and 52-56 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, R. flavipes, or Neosartorya fischeri cellobiohydrolase, endoglucanase or beta-glucosidase polypeptide sequences: amino acid sequence variants of such sequences wherein at least one amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequences, or their fragments as defined above, that have been substituted by another residue. Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to bacterial, fungal, insect, rabbit, rat, porcine, bovine, ovine, equine and non-human primate species, the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).

[0148] Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the cellulase polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.

[0149] Thus, the invention further includes H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.

[0150] The skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.

[0151] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

[0152] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

[0153] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

[0154] As the authors state, these two strategies have revealed that proteins are often surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

[0155] The terms "derivative" and "analog" refer to a polypeptide differing from the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptide, but retaining essential properties thereof. Generally, derivatives and analogs are overall closely similar, and, in many regions, identical to the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridium thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides. The terms "derivative" and "analog" when referring to H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides include any polypeptides which retain at least some of the activity of the corresponding native polypeptide, e.g., the exoglucanase activity, or the activity of the catalytic domain.

[0156] Derivatives of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Derivatives can be covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope). Examples of derivatives include fusion proteins.

[0157] An analog is another form of a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellobiohydrolase, endoglucanase or beta-glucosidase polypeptide of the present invention. An "analog" also retains substantially the same biological function or activity as the polypeptide of interest, e.g., functions as a cellobiohydrolase. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

[0158] The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In some particular embodiments, the polypeptide is a recombinant polypeptide.

[0159] Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to any of SEQ ID NOs: 1-40, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

Consensus Sequence Cellulases

[0160] In some embodiments of the present invention, the host cells express at least one heterologous cellulase that is not derived from any one particular organism, but instead has an artificial amino acid sequence that is a consensus cellulase sequence. The consensus cellulase sequence can be an endoglucanase consensus sequence, a .beta.-glucosidase consensus sequence, or a cellobiohydrolase consensus sequence.

[0161] In one particular embodiment, the heterologous cellulase is a CBH1 consensus sequence. Therefore, in one embodiment, the invention is directed to a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the consensus CBH1 sequence of SEQ ID NO: 43. In some embodiments, the invention is directed to a polypeptide which comprises the sequence of SEQ ID NO: 43.

[0162] The invention is also directed to host cells that comprise a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% 99% or 100% identical to the consensus CBH1 sequence of SEQ ID NO: 43. The invention further directed to host cells that comprise a polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% 99% or 100% identical to the consensus CBH1 sequence of SEQ ID NO: 43. In some embodiments the host cell comprises at least one polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the consensus CBH1 sequence of SEQ ID NO: 43 and at least a second polynucleotide that encodes a heterologous cellulase. The second polynucleotide can encode a endoglucanase, a .beta.-glucosidase, a cellobiohydrolase, an endoglucanase consensus sequence, a .beta.-glucosidase consensus sequence, or a cellobiohydrolase consensus sequence. In some embodiments the host cell comprising the polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the consensus CBH1 sequence of SEQ ID NO: 43 is capable of producing ethanol when grown using cellulose as a carbon source.

Combinations of Cellulases

[0163] In some embodiments of the present invention the host cells express a combination of heterologous cellulases. For example, the host cell can contain at least two heterologous cellulases, at least three heterologous cellulases, at least four heterologous cellulases, at least five heterologous cellulases, at least six heterologous cellulases, at least seven heterologous cellulases, at least eight heterologous cellulases, at least nine heterologous cellulases, at least ten heterologous cellulases, at least eleven heterologous cellulases, at least twelve heterologous cellulases, at least thirteen heterologous cellulases, at least fourteen heterologous cellulases or at least fifteen heterologous cellulases. The heterologous cellulases in the host cell can be from the same or from different species.

[0164] In some embodiments of the present invention, the host cells express a combination of heterologous cellulases which includes at least one endoglucanase, at least one .beta.-glucosidase and at least one cellobiohydrolase. In another embodiment of the invention, the host cells express a combination of heterologous cellulases which includes at least one endoglucanase, at least one .beta.-glucosidase and at least two cellobiohydrolases. The at least two cellobiohydrolases can be both be cellobiohydrolase I, can both be cellobiohydrolase II, or can be one cellobiohydrolase I and one cellobiohydrolase II.

[0165] In one particular embodiment of the invention, the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I and an S. fibuligera .beta.-glucosidase I. In another embodiment of the invention, the host cells express a combination of cellulases that includes a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II.

[0166] In yet another embodiment the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I, an S. fibuligera .beta.-glucosidase I, a T. emersonii cellobiohydrolase I, and a C. lucknowense cellobiohydrolase IIb. In still another embodiment, the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I, an S. fibuligera .beta.-glucosidase I, a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II. In still another embodiment, the host cells express a combination of cellulases that includes an H. jecorina endogluconase 2, an S. fibuligera .beta.-glucosidase I, a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II. In still another embodiment, the host cells express a combination of cellulases that includes an H. jecorina endogluconase 2, an S. fibuligera .beta.-glucosidase I, a T. emersonii cellobiohydrolase I, and a C. lucknowense cellobiohydrolase II.

Tethered and Secreted Cellulases

[0167] According to the present invention, the cellulases may be either tethered or secreted. As used herein, a protein is "tethered" to an organism's cell surface if at least one terminus of the protein is bound, covalently and/or electrostatically for example, to the cell membrane or cell wall. It will be appreciated that a tethered protein may include one or more enzymatic regions that may be joined to one or more other types of regions at the nucleic acid and/or protein levels (e.g., a promoter, a terminator, an anchoring domain, a linker, a signaling region, etc.). While the one or more enzymatic regions may not be directly bound to the cell membrane or cell wall (e.g., such as when binding occurs via an anchoring domain), the protein is nonetheless considered a "tethered enzyme" according to the present specification.

[0168] Tethering may, for example, be accomplished by incorporation of an anchoring domain into a recombinant protein that is heterologously expressed by a cell, or by prenylation, fatty acyl linkage, glycosyl phosphatidyl inositol anchors or other suitable molecular anchors which may anchor the tethered protein to the cell membrane or cell wall of the host cell. A tethered protein maybe tethered at its amino terminal end or optionally at its carboxy terminal end.

[0169] As used herein, "secreted" means released into the extracellular milieu, for example into the media. Although tethered proteins may have secretion signals as part of their immature amino acid sequence, they are maintained as attached to the cell surface, and do not fall within the scope of secreted proteins as used herein.

[0170] As used herein, "flexible linker sequence" refers to an amino acid sequence which links two amino acid sequences, for example, a cell wall anchoring amino acid sequence with an amino acid sequence that contains the desired enzymatic activity. The flexible linker sequence allows for necessary freedom for the amino acid sequence that contains the desired enzymatic activity to have reduced steric hindrance with respect to proximity to the cell and may also facilitate proper folding of the amino acid sequence that contains the desired enzymatic activity.

[0171] In some embodiments of the present invention, the tethered cellulase enzymes are tethered by a flexible linker sequence linked to an anchoring domain. In some embodiments, the anchoring domain is of CWP2 (for carboxy terminal anchoring) or FLO1 (for amino terminal anchoring) from S. cerevisiae.

[0172] In some embodiments, heterologous secretion signals may be added to the expression vectors of the present invention to facilitate the extra-cellular expression of cellulase proteins. In some embodiments, the heterologous secretion signal is the secretion signal from T. reesei Xyn2.

Fusion Proteins Comprising Cellulases

[0173] The present invention also encompasses fusion proteins. For example, the fusion proteins can be a fusion of a heterologous cellulase and a second peptide. The heterologous cellulase and the second peptide can be fused directly or indirectly, for example, through a linker sequence. The fusion protein can comprise for example, a second peptide that is N-terminal to the heterologous cellulase and/or a second peptide that is C-terminal to the heterologous cellulase. Thus, in certain embodiments, the polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a heterologous cellulase.

[0174] According to the present invention, the fusion protein can comprise a first and second polypeptide wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a signal sequence. According to another embodiment, the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a polypeptide used to facilitate purification or identification or a reporter peptide. The polypeptide used to facilitate purification or identification or the reporter peptide can be, for example, a HIS-tag, a GST-tag, an HA-tag, a FLAG-tag, a MYC-tag, or a fluorescent protein.

[0175] According to yet another embodiment, the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises an anchoring peptide. In some embodiments, the anchoring domain is of CWP2 (for carboxy terminal anchoring) or FLO1 (for amino terminal anchoring) from S. cerevisiae.

[0176] According to yet another embodiment, the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a cellulose binding module (CBM). In some embodiments, the CBM is from, for example, T. reesei Cbh1 or Cbh2, from H. grisea Cbh1, or from C. lucknowense Cbh2b. In some particular embodiments, the CBM is fused to a cellobiohydrolase. In one particular embodiment, the fusion protein comprises a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellobiohydrolase and the second polypeptide comprises a CBM. In yet another particular embodiment, the cellobiohydrolase is T. emersonii cellobiohydrolase I and the CBM is a T. reesei cellobiohydrolase CBM. In yet another particular embodiment, the cellobiohydrolase is T. emersonii cellobiohydrolase I and the CBM is a H. grisea cellobiohydrolase CBM. In some embodiments, the CBM of H. grisea comprises amino acids 492-525 of SEQ ID NO: 21.

[0177] In certain embodiments, the polypeptide of the present invention encompasses a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a cellobiohydrolase, and the second polypeptide is a domain or fragment of a cellobiohydrolase. In certain embodiments, the polypeptide of the present invention encompasses a fusion protein comprising a first polypeptide, where the first polypeptide is a T. emersonii Cbh1, H. grisea Cbh1, T. aurantiacusi Cbh1, T. emersonii Cbh2, T. reesei Cbh1 T. reesei Cbh2, C. lucknowense Cbh2b, or domain, fragment, variant, or derivative thereof, and a second polypeptide, where the second polypeptide is a T. emersonii Cbh1, H. grisea Cbh1, or T. aurantiacusi Cbh1, T. emersonii Cbh2, T. reesei Cbh1 or T. reesei Cbh2, C. lucknowense Cbh2b, or domain, fragment, variant, or derivative thereof. In particular embodiments the first polypeptide is T. emersonii Cbh1 and the second polynucleotide is a CBM from T. reesei Cbh1 or Cbh2 or from C. lucknowense Cbh2b. In additional embodiments, the first polypeptide is either N-terminal or C-terminal to the second polypeptide. In certain other embodiments, the first polypeptide and/or the second polypeptide are encoded by codon-optimized polynucleotides, for example, polynucleotides codon-optimized for S. cerevisiae or Kluyveromyces. In particular embodiments, the first polynucleotide is a codon-optimized T. emersonii cbh1 and the second polynucleotide encodes for a codon-optimized CBM from T. reesei Cbh1 or Cbh2. In another particular embodiments, the first polynucleotide is a codon-optimized T. emersonii cbh1 and the second polynucleotide encodes for a codon-optimized CBM from C. lucknowense or Cbh2b.

[0178] In certain other embodiments, the first polypeptide and the second polypeptide are fused via a linker sequence. The linker sequence can, in some embodiments, be encoded by a codon-optimized polynucleotide. (Codon-optimized polynucleotides are described in more detail below.) An amino acid sequence corresponding to a codon-optimized linker 1 according to the invention is a flexible linker-strep tag-TEV site-FLAG-flexible linker fusion and corresponds to GGGGSGGGGS AWHPQFGG ENLYFQG DYKDDDK GGGGSGGGGS (SEQ ID NO:57)

[0179] The DNA sequence is as follows:

TABLE-US-00002 (SEQ ID NO: 41) GGAGGAGGTGGTTCAGGAGGTGGTGGGTCTGCTTGGCAT CCACAATTTGGAGGAGGCGGTGGTGAAAATCTGTATTTC CAGGGAGGCGGAGGTGATTACAAGGATGACGACAAAGG AGGTGGTGGATCAGGAGGTGGTGGCTCC

[0180] An amino acid sequence corresponding to optimized linker 2 is a flexible linker-strep tag-linker-TEV site-flexible linker and corresponds to GGGGSGGGGS WSHPQFEK GG ENLYFQG GGGGSGGGGS (SEQ ID NO:58). The DNA sequence is as follows:

TABLE-US-00003 (SEQ ID NO: 42) ggtggcggtggatctggaggaggcggttcttggtctcacccacaatttga aaagggtggagaaaacttgtactttcaaggcggtggtggaggttctggcg gaggtggctccggctca

Co-Cultures

[0181] The present invention is also directed to co-cultures comprising at least two yeast host cells wherein the at least two yeast host cells each comprise an isolated polynucleotide encoding a heterologous cellulase. As used herein. "co-culture" refers to growing two different strains or species of host cells together in the same vessel. In some embodiments of the invention, at least one host cell of the co-culture comprises a heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase, at least one host cell of the co-culture comprises a heterologous polynucleotide comprising a nucleic acid which encodes a .beta.-glucosidase and at least one host cell comprises a heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase. In a further embodiment, the co-culture further comprises a host cell comprising a heterologous polynucleotide comprising a nucleic acid which encodes a second cellobiohydrolase.

[0182] The co-culture can comprise two or more strains of yeast host cells and the heterologous cellulases can be expressed in any combination in the two or more strains of host cells. For example, according to the present invention, the co-culture can comprise two strains: one strain of host cells that expresses an endoglucanase and a second strain of host cells that expresses a .beta.-glucosidase, a cellobiohydrolase and a second cellobiohydrolase. According to the present invention, the co-culture can also comprise four strains: one strain of host cells which expresses an endoglucanase, one strain of host cells that expresses a .beta.-glucosidase, one strain of host cells which expresses a first cellobiohydrolase, and one strain of host cells which expresses a second cellobiohydrolase. Similarly, the co-culture can comprise one strain of host cells that expresses two cellulases, for example an endoglucanase and a beta-glucosidase and a second strain of host cells that expresses one or more cellulases, for example one or more cellobiohydrolases. The co-culture can, in addition to the at least two host cells comprising heterologous cellulases, also include other host cells which do not comprise heterologous cellulases.

[0183] The various host cell strains in the co-culture can be present in equal numbers, or one strain or species of host cell can significantly outnumber another second strain or species of host cells. For example, in a co-culture comprising two strains or species of host cells the ratio of one host cell to another can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:100, 1:500 or 1:1000. Similarly, in a co-culture comprising three or more strains or species of host cells, the strains or species of host cells may be present in equal or unequal numbers.

[0184] The co-cultures of the present invention can include tethered cellulases, secreted cellulases or both tethered and secreted cellulases. For example, in some embodiments of the invention, the co-culture comprises at least one yeast host cell comprising a polynucleotide encoding a secreted heterologous cellulase. In another embodiment, the co-culture comprises at least one yeast host cell comprising a polynucleotide encoding a tethered heterologous cellulase. In one embodiment, all of the heterologous cellulases in the co-culture are secreted, and in another embodiment, all of the heterologous cellulases in the co-culture are tethered. In addition, other cellulases, such as externally added cellulases may be present in the co-culture.

Polynucleotides Encoding Heterologous Cellulases

[0185] The present invention also includes isolated polynucleotides encoding cellulases of the present invention. Thus, the polynucleotides of the invention can encode endoglucanases or exoglucanases. The polynucleotides can encode endoglucanases, .beta.-glucosidases or cellobiohydrolases.

[0186] In some particular embodiments of the invention, the polynucleotide encodes an endoglucanase which is an endo-1,4-.beta.-glucanase. In particular embodiments, the polynucleotide encodes an endoglucanase I from Trichoderma reesei. In certain other embodiments, the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:19. In particular embodiments, the polynucleotide encodes an endoglucanase I from C. formosanus. In certain other embodiments, the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:11. In particular embodiments, the polynucleotide encodes an endoglucanase I from Trichoderma reesei. In certain other embodiments, the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:19. In particular embodiments, the polynucleotide encodes an endoglucanase 2 from H. jecorina. In certain other embodiments, the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:54.

[0187] In certain embodiments, the polynucleotide encodes a .beta.-glucosidase I or a .beta.-glucosidase II isoform, paralogue or orthologue. In certain embodiments of the present invention the polynucleotide encodes a .beta.-glucosidase derived from Saccharomycopsis fibuligera. In particular embodiments, the .beta.-glucosidase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:20.

[0188] In certain embodiments of the invention, the polynucleotide encodes a cellobiohydrolase I and/or an cellobiohydrolase II isoform, paralogue or orthologue. In particular embodiments of the present invention, the polynucleotide encodes the cellobiohydrolase I or II from Trichoderma reesei. In particular embodiments of the present invention, the polynucleotide encodes the cellobiohydrolase I or II from Trichoderma emersonii. In another embodiment, the cellobiohydrolase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:7 or SEQ ID NO:8. In particular embodiments of the present invention, the polynucleotide encodes a cellobiohydrolase from C. lucknowense. In another embodiment, the cellobiohydrolase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:5.

[0189] In further embodiments the polynucleotide is a polypeptide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to a nucleotide sequence listed in Table 1. In certain aspects the polynucleotide can encode an endoglucanase, cellobiohydrolase or .beta.-glucosidase derived from, for example, a fungal, bacterial, protozoan or termite source.

[0190] In certain aspects, the present invention relates to a polynucleotide comprising a nucleic acid encoding a functional or structural domain of T. emersonii, H. grisea, T. aurantiacus, C. lucknowense or T. reesei Cbh1 or Cbh2. For example, the domains of T. reesei Cbh1 include, without limitation: (1) a signal sequence, from amino acid 1 to 33 of SEQ ID NO: 27; (2) a catalytic domain (CD) from about amino acid 41 to about amino acid 465 of SEQ ID NO: 27; and (3) a cellulose binding module (CBM) from about amino acid 503 to about amino acid 535 of SEQ ID NO: 27. The domains of T. reesei Cbh 2 include, without limitation: (1) a signal sequence, front amino acid 1 to 33 of SEQ ID NO: 27; (2) a catalytic domain (CD) from about amino acid 145 to about amino acid 458 of SEQ ID NO: 27; and (3) a cellulose binding module (CBM) from about amino acid 52 to about amino acid 83 of SEQ ID NO: 27.

[0191] The present invention also encompasses an isolated polynucleotide comprising a nucleic acid that is at least about 70%, 75%, or 80% identical, at least about 90% to about 95% identical, or at least about 96%, 97%, 98%, 99% or 100% identical to a nucleic acid encoding a T. emersonii, H. grisea. T. aurantiacus, C. lucknowense or T. reesei Cbh1 or Cbh2 domain, as described above.

[0192] The present invention also encompasses variants of the cellulase genes, as described above. Variants may contain alterations in the coding regions, non-coding regions, or both. Examples are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In certain embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In further embodiments, H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellulase polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host. Codon-optimized polynucleotides of the present invention are discussed further below.

[0193] The present invention also encompasses an isolated polynucleotide encoding a fusion protein. In certain embodiments, the nucleic acid encoding a fusion protein comprises a first polynucleotide encoding for a T. emersonii cbh1, H. grisea cbh1, T. aurantiancusi cbh1 or T. emersonii cbh1 and a second polynucleotide encoding for the CBM domain of T. reesei cbh1 or T. reesei cbh2 or C. lucknowense cbh2b. In particular embodiments of the nucleic acid encoding a fusion protein, the first polynucleotide encodes T. emersonii cbh1 and the second polynucleotide encodes for a CBM from T. reesei Cbh1 or Cbh2.

[0194] In further embodiments, the first and second polynucleotides are in the same orientation, or the second polynucleotide is in the reverse orientation of the first polynucleotide. In additional embodiments, the first polynucleotide encodes a polypeptide that is either N-terminal or C-terminal to the polypeptide encoded by the second polynucleotide. In certain other embodiments, the first polynucleotide and/or the second polynucleotide are encoded by codon-optimized polynucleotides, for example, polynucleotides codon-optimized for S. cerevisiae, Kluyveromyces or for both S. cerevisiae and Kluyveromyces. In particular embodiments of the nucleic acid encoding a fusion protein, the first polynucleotide is a codon-optimized T. emersonii cbh1 and the second polynucleotide encodes for a codon-optimized CBM from T. reesei Cbh1 or Cbh2.

[0195] Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to any of SEQ ID NOs: 1-20, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

[0196] By a nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the particular polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown of any of SEQ ID NOs:1-20, or any fragment or domain specified as described herein.

[0197] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence or polypeptide of the present invention can be determined conventionally using known computer programs. A method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

[0198] If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

[0199] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.

[0200] Some embodiments of the invention encompass a nucleic acid molecule comprising at least 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, or 800 consecutive nucleotides or more of any of SEQ ID NOs: 1-20, or domains, fragments, variants, or derivatives thereof.

[0201] The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide can be identical to the coding sequence encoding SEQ ID NO:21-40, 46, or 52-56, or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of any one of SEQ ID NOs:21-40, 46, or 52-56.

[0202] In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleic acid fragment which encodes at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, or at least 100 or more contiguous amino acids of SEQ ID NOs: 21-40, 46, or 52-56.

[0203] The polynucleotide encoding for the mature polypeptide of SEQ ID NOs: 21-40, 46, or 52-56 or may include: only the coding sequence for the mature polypeptide; the coding sequence of any domain of the mature polypeptide; and the coding sequence for the mature polypeptide (or domain-encoding sequence) together with non coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

[0204] Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only sequences encoding for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.

[0205] In further aspects of the invention, nucleic acid molecules having sequences at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein, encode a polypeptide having cellobiohydrolase ("Cbh"), endoglucanase ("Eg") or beta-gluconase ("Bgl") functional activity. By "a polypeptide having Cbh, Eg or Bgl functional activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the Cbh, Eg or Bgl polypeptides of the present invention, as measured, for example, in a particular biological assay. For example, a Cbh, Eg or Bgl functional activity can routinely be measured by determining the ability of a Cbh, Eg or Bgl polypeptide to hydrolyze cellulose, or by measuring the level of Cbh, Eg or Bgl activity.

[0206] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large portion of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 1-20, or fragments thereof, will encode polypeptides having Cbh, Eg or Bgl functional activity. In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having Cbh, Eg or Bgl functional activity.

[0207] The polynucleotides of the present invention also comprise nucleic acids encoding a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellulase, or domain, fragment, variant, or derivative thereof, fused to a polynucleotide encoding a marker sequence which allows for detection of the polynucleotide of the present invention. In one embodiment of the invention, expression of the marker is independent from expression of the cellulase. The marker sequence may be a yeast selectable marker selected from the group consisting of URA3, HIS3, LEU2, TRP1, LYS2 or ADE2. Casey, G. P. et al., "A convenient dominant selection marker for gene transfer in industrial strains of Saccharomyces yeast: SMR1 encoded resistance to the herbicide sulfometuron methyl," J. Inst. Brew. 94:93-97 (1988).

Codon Optimized Polynucleotides

[0208] According to one embodiment of the invention, the polynucleotides encoding heterologous cellulases can be codon-optimized. As used herein the term "codon-optimized coding region" means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

[0209] In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the "codon adaptation index" or "CAI," which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism.

[0210] The CAI of codon optimized sequences of the present invention corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0. A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of "As" or "Ts" (e.g., runs greater than 4, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can be removed from the sequences if these are known to effect transcription negatively. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes. Examples of such restriction enzyme sites include PacI, AscI, BamHI, BglII, EcoRI and XhoI. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with "second best" codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized.

[0211] Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is reproduced herein as Table 2. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

TABLE-US-00004 TABLE 2 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC '' TCC '' TAC '' TGC TTA Leu (L) TCA '' TAA Ter TGA Ter TTG '' TCG '' TAG Ter TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC '' CCC '' CAC '' GCG '' CTA '' CCA '' CAA Gln (Q) CGA '' CTG '' CCG '' CAG '' CGG '' A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC '' ACC '' AAC '' AGC '' ATA '' ACA '' AAA Lys (K) AGA Arg (R) ATG Met (M) ACG '' AAG '' AGG '' G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC '' GCC '' GAC '' GGC '' GTA '' GCA '' GAA Glu (E) GGA '' GTG '' GCG '' GAG '' GGG ''

[0212] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0213] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at http://phenotype.biosci.umbe.edu/codon/sgd/index.php (visited May 7, 2008) or at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 3. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The Table has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

TABLE-US-00005 TABLE 3 Codon Usage Table for Saccharomyces cerevisicie Genes Amino Frequency per Acid Codon Number hundred Phe UUU 170666 26.1 Phe UUC 120510 18.4 Total Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC 35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Total

[0214] By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. Codon-optimized coding regions can be designed by various different methods.

[0215] In one method, a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 3 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG.

[0216] In another method, the actual frequencies of the codons are distributed randomly throughout the coding sequence. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 1000 leucine residues, referring to Table 3 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.

[0217] These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method; however, the sequence always encodes the same polypeptide.

[0218] When using the methods above, the term "about" is used precisely to account for fractional percentages of codon frequencies for a given amino acid. As used herein, "about" is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less. Using again the example of the frequency of usage of leucine in human genes for a hypothetical polypeptide having 62 leucine residues, the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or "about 5," i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 UUG codons or "about 8," i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or "about 8," i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or "about 12," i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or "about 4," i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or "about 25," i.e., 24, 25, or 26 CUG codons.

[0219] Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the "backtranslate" function in the GCG--Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the "backtranslation" function at http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visited Apr. 15, 2008) and the "backtranseq" function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.

[0220] A number of options are available for synthesizing codon optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence is synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO.RTM. vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.

[0221] In certain embodiments, an entire polypeptide sequence, or fragment, variant, or derivative thereof is codon optimized by any of the methods described herein. Various desired fragments, variants or derivatives are designed, and each is then codon-optimized individually. In addition, partially codon-optimized coding regions of the present invention can be designed and constructed. For example, the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, or (of the codon positions have been codon-optimized for a given species. That is, they contain a codon that is preferentially used in the genes of a desired species, e.g., a yeast species such as Saccharomyces cerevisiae or Kluyveromyces, in place of a codon that is normally used in the native nucleic acid sequence.

[0222] In additional embodiments, a full-length polypeptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire polypeptide, and then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide are made from the original codon-optimized coding region. As would be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advantage of this approach is that synthesizing codon-optimized nucleic acid fragments encoding each fragment, variant, and derivative of a given polypeptide, although routine, would be time consuming and would result in significant expense.

[0223] The codon-optimized coding regions can be, for example, versions encoding a cellobiohydrolase, endoglucanase or beta-glucosidase from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinonmyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana or domains, fragments, variants, or derivatives thereof.

[0224] Codon optimization is carried out for a particular species by methods described herein, for example, in certain embodiments codon-optimized coding regions encoding polypeptides of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinonmyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes, or Arabidopsis thaliana cellulases, or domains, fragments, variants, or derivatives thereof are optimized according to yeast codon usage, e.g., Saccharomyces cerevisiae, Kluyveromyces lactis and/or Kluyveromyces marxianus. Also provided are polynucleotides, vectors, and other expression constructs comprising codon-optimized coding regions encoding polypeptides of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, R. flavipes or Arabidopsis thaliana cellulases or domains, fragments, variants, or derivatives thereof, and various methods of using such polynucleotides, vectors and other expression constructs.

[0225] In certain embodiments described herein, a codon-optimized coding region encoding any of SEQ ID NOs:21-40, 46, or 52-56 or domain, fragment, variant, or derivative thereof, is optimized according to codon usage in yeast (Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxianus). In some embodiments, the sequences are codon-optimized specifically for expression in Saccharomyces cerevisiae. In some embodiments, the sequences are codon-optimized for expression in Kluyveromyces. In some embodiments, a sequence is simultaneously codon-optimized for optimal expression in both Saccharomyces cerevisiae and in Kluyveromyces. Alternatively, a codon-optimized coding region encoding any of SEQ ID NOs: 21-40, 46, or 52-56 may be optimized according to codon usage in any plant, animal, or microbial species.

Vectors and Methods of Using Vectors in Host Cells

[0226] The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

[0227] Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0228] The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids: and yeast plasmids. However, any other vector may be used as long as it is replicable and viable in the host.

[0229] The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0230] The DNA sequence in the expression vector is operatively associated with an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Representative examples of such promoters are as follows:

TABLE-US-00006 Gene Organism Systematic name Reason for use/benefits PGK1 S. cerevisiae YCR012W Strong constitutive promoter ENO1 S. cerevisiae YGR254W Strong constitutive promoter TDH3 S. cerevisiae YGR192C Strong constitutive promoter TDH2 S. cerevisiae YJR009C Strong constitutive promoter TDH1 S. cerevisiae YJL052W Strong constitutive promoter ENO2 S. cerevisiae YHR174W Strong constitutive promoter GPM1 S. cerevisiae YKL152C Strong constitutive promoter TPI1 S. cerevisiae YDR050C Strong constitutive promoter

[0231] Additionally, promoter sequences from stress and starvation response genes are useful in the present invention. In some embodiments, promoter regions from the S. cerevisiae genes GAC1, GET3, GLC7, GSH1, GSH2, HSF1, HSP12, LCB5, LRE1, LSP1, NBP2, PIL1, PIM1, SGT2, SLG1, WHI2, WSC2, WSC3, WSC4, YAP1, YDC1, HSP104, HSP26, ENA1, MSN2, MSN4, SIP2, SIP4, SIP5, DPL1, IRS4, KOG1, PEP4, HAP4, PRB1, TAX4, ZPR1, ATG1, ATG2, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, and ATG19 may be used. Any suitable promoter to drive gene expression in the host cells of the invention may be used. Additionally the E. coli, lac or trp, and other promoters known to control expression of genes in prokaryotic or lower eukaryotic cells can be used.

[0232] In addition, the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as URA3, HIS3, LEU2, TRP1, LYS2 or ADE2, dihydrofolate reductase, neomycin (G418) resistance or zeocin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.

[0233] The expression vector may also contain a ribosome binding site for translation initiation and/or a transcription terminator. The vector may also include appropriate sequences for amplifying expression, or may include additional regulatory regions.

[0234] The vector containing the appropriate DNA sequence as herein, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

[0235] Thus, in certain aspects, the present invention relates to host cells containing the above-described constructs. The host cell can be a host cell as described elsewhere in the application. The host cell can be, for example, a lower eukaryotic cell, such as a yeast cell, e.g., Saccharomyces cerevisiae or Klyuveromyces, or the host cell can be a prokaryotic cell, such as a bacterial cell.

[0236] As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; thermophilic or mesophlic bacteria; fungal cells, such as yeast; and plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0237] Appropriate fungal hosts include yeast. In certain aspects of the invention the yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Issatchenkia orientalis, Kluyveromyces marxianus, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia.

Methods of Using Host Cells to Produce Ethanol

[0238] The present invention is also directed to use of host cells and co-cultures to produce ethanol from cellulosic substrates. Such methods can be accomplished, for example, by contacting a cellulosic substrate with a host cell or a co-culture of the present invention.

[0239] Numerous cellulosic substrates can be used in accordance with the present invention. Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

[0240] It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.

[0241] In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a host cell of the invention. In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a co-culture comprising yeast cells expressing heterologous cellulases.

[0242] In some embodiments, the invention is directed to a method for fermenting cellulose. Such methods can be accomplished, for example, by culturing a host cell or co-culture in a medium that contains insoluble cellulose to allow saccharification and fermentation of the cellulose.

[0243] The production of ethanol can, according to the present invention, be performed at temperatures of at least about 30.degree. C., about 31.degree. C., about 32.degree. C., about 33.degree. C., about 34.degree. C., about 35.degree. C., about 36.degree. C., about 37.degree. C., about 38.degree. C., about 39.degree. C., about 40.degree. C., about 41.degree. C., about 42.degree. C., about 43.degree. C., about 44.degree. C., about 45.degree. C., about 46.degree. C., about 47.degree. C., about 48.degree. C., about 49.degree. C., or about 50.degree. C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol front cellulose at temperatures above about 30.degree. C., about 31.degree. C., about 32.degree. C., about 33.degree. C., about 34.degree. C., about 35.degree. C., about 36.degree. C., about 37.degree. C., about 38.degree. C., about 39.degree. C. about 40.degree. C., about 41.degree. C., about 42.degree. C., or about 43.degree. .degree. C., or about 44.degree. C., or about 45.degree. C., or about 50.degree. C. In some embodiments of the present invention, the thermotolerant host cell can produce ethanol from cellulose at temperatures from about 30.degree. C. to 60.degree. C., about 30.degree. C. to 55.degree. .degree. C., about 30.degree. C. to 50.degree. C., about 40.degree. C. to 60.degree. C., about 40.degree. C. to 55.degree. C. or about 40.degree. C. to 50.degree. C.

[0244] In some embodiments, methods of producing ethanol can comprise contacting a cellulosic substrate with a host cell or co-culture of the invention and additionally contacting the cellulosic substrate with externally produced cellulase enzymes. Exemplary externally produced cellulase enzymes are commercially available and are known to those of skill in the art.

[0245] Therefore, the invention is also directed to methods of reducing the amount of externally produced cellulase enzymes required to produce a given amount of ethanol from cellulose comprising contacting the cellulose with externally produced cellulases and with a host cell or co-culture of the invention. In some embodiments, the same amount of ethanol production can be achieved using at least about 5%, 10%, 15%, 20%, 25%, 30%, or 50% less externally produced cellulases. In some embodiments, no external cellulase is added, or less than about 5% of the cellulase is externally added cellulase, or less than about 10% of the cellulase is externally added cellulase, or less than about 15% of the cellulase is externally added cellulase.

[0246] In some embodiments, the methods comprise producing ethanol at a particular rate. For example, in some embodiments, ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter.

[0247] In some embodiments, the host cells of the present invention can produce ethanol at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter more than a control strain (lacking heterologous cellulases) and grown under the same conditions. In some embodiments, the ethanol can be produced in the absence of any externally added cellulases.

[0248] Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays. Methods of determining ethanol production are within the scope of those skilled in the art from the teachings herein.

[0249] The following embodiments of the invention will now be described in more detail by way of these non-limiting examples.

EXAMPLES

[0250] The present invention presents a number of important steps forward for creating a yeast capable of consolidated bioprocessing. It describes improved cellulolytic yeast created by expressing combinations of heterologous cellulases. The present invention demonstrates for the first time, the ability of transformed Kluyveromyces to produce ethanol from cellulose, the ability of yeast strains expressing only secreted heterologous cellulases to produce ethanol from cellulose, and the ability of co-cultures of multiple yeast strains expressing different cellulases to produce ethanol from cellulose. In addition such yeast strains and co-cultures of yeast strains can increase the efficiency of simultaneous saccharification and fermentation (SSF) processes.

General Protocols

[0251] General Strain Cultivation and Media

[0252] Escherichia coli strain DH5a (Invitrogen), or NEB 5 alpha (New England Biolabs) was used for plasmid transformation and propagation. Cells were grown in LB medium (5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone) supplemented with ampicillin (100 mg/L), kanamycin (50 mg/L), or zeocin (20 mg/L). When zeocin selection was desired LB was adjusted to pH 7.0. Also, 15 g/L agar was added when solid media was desired.

[0253] Yeast strains were routinely grown in YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose), YPC (10 g/L yeast extract, 20 g/L peptone, 20 g/L cellobiose), or YNB+glucose (6.7 g/L Yeast Nitrogen Base without amino acids, and supplemented with appropriate amino acids for strain, 20 g/L glucose) media with either G418 (250 mg/L unless specified) or zeocin (20 mg/L unless specified) for selection. 15 g/L agar was added for solid media.

[0254] Molecular Methods

[0255] Standard protocols were followed for DNA manipulations (Sambrook et al. 1989). PCR was performed using Phusion polymerase (New England Biolabs) for cloning, and Taq polymerase (New England Biolabs) for screening transformants, and in some cases Advantage Polymerase (Clontech) for PCR of genes for correcting auxotrophies. Manufacturers guidelines were followed as supplied. Restriction enzymes were purchased from New England Biolabs and digests were set up according to the supplied guidelines. Ligations were performed using the Quick ligation kit (New England Biolabs) as specified by the manufacturer. Gel purification was performed using either Qiagen or Zymo research kits, PCR product and digest purifications were performed using Zymo research kits, and Qiagen midi and miniprep kits were used for purification of plasmid DNA. Sequencing was performed by the Molecular Biology Core Facility at Dartmouth College. Yeast mediated ligation (YML) was used to create some constructs (Ma et al. Gene 58:201-216 (1987)). This was done by creating DNA fragments to be cloned with 20-40 bp of homology with the other pieces to be combined and/or the backbone vector. A backbone vector (pRS426), able to replicate in yeast, and with the Ura3 gene for selection, was then transformed into yeast by standard methods with the target sequences for cloning. Transformed yeast recombine these fragments to form a whole construct and the resulting plasmid allows selection on media without uracil.

[0256] Vectors

[0257] Plasmid constructs vectors in the experiments detailed below are summarized in Table 4, and the printers used in vector construction are shown in Table 5.

TABLE-US-00007 TABLE 4 Plasmids used. Plasmid Genotype pBKD1-BGLI bla KanMX PGK1.sub.P-S.f. bgl1- PGK1.sub.T pBKD2-sEGI bla KanMX ENO1.sub.P-sT.r. eg1- ENO1.sub.T pBKD1-BGLI-sEGI bla KanMX ENO1.sub.P-sT.r. eg1- ENO1.sub.T & PGK1.sub.P-S.f. bgl1- PGK1.sub.T YEpENO-BBH bla URA3 ENO1.sub.PT pJC1 La grange et al. bla URA3 PGK.sub.PT (1996) pRDH101 bla URA3 ENO1.sub.P-sT.r.cbh1- ENO1.sub.T pRDH103 bla URA3 ENO1.sub.P-sH.g.cbh1- ENO1.sub.T pRDH104 bla URA3 ENO1.sub.P-sT.a.cbh1- ENO1.sub.T pRDH105 bla URA3 ENO1.sub.P-sT.e.cbh1- ENO1.sub.T pRDH106 bla URA3 ENO1.sub.P-sT.e.cbh2- ENO1.sub.T pRDH107 bla URA3 PGK1.sub.P-sT.r.cbh2- PGK1.sub.T pRDH108 bla URA3 PGK1.sub.P-sT.r.cbh2- PGK1.sub.T & ENO1.sub.P-sT.e.cbh1- ENO1.sub.T pRDH118 bla URA3 PGK1.sub.P-sT.r.cbh2- PGK1.sub.T & ENO1.sub.P-sH.g.cbh1- ENO1.sub.T pRDH120 bla URA3 PGK1.sub.P-sT.r.cbh2- PGK1.sub.T & ENO1.sub.P-sT.a.cbh1- ENO1.sub.T pDF1 La Grange et al. bla fur1::LEU2 (1996) pCEL5 Den Haan et al. 2 micron vector for expression of SfBGLI and 2007 TrEGI (native sequence) pMU185 pUG66 (loxp-zeo-loxp) pKLAC1 New England K. lactis expression vector for integration at Biolabs the lac4 locus, acetamide selection pRS426 2 micron vector for yeast mediated ligation (YML) pMU289 pRS426 with portion of pKLAC1 for insertion of TrEG1 (from pBKD_11621, as detailed in example 1) into lac4 locus created by YML pMU291 pRS426 with portion of pKLAC1 for insertion of TrCBH2 (from pBZD_20641, as detailed in example 1) into lac4 locus created by YML pMU398 ENO1.sub.P-sT.e.cbh1- ENO1.sub.T from pRDH105 into pMU289 (cloning by YML) pMU451 pRDH105 with PacI/AscI linker (formed using primers) inserted into EcoRI/XhoI pMU458 synthetic construct for N.f. EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU463 TrEG1 from pBKD1-BGLI-sEGI into pMU451 (PacI/AscI digest of both pieces) pMU465 synthetic construct for C.l.(a) EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU469 synthetic construct for R.f.EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU471 synthetic construct for C.f.EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU472 synthetic construct for N.t.EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU473 synthetic construct for C.a.EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU475 synthetic construct for T.r. CBH2 derived from pBKD_20641 with tether removed (from example 1) inserted into pMU451 (PacI/AscI digest of both pieces) pMU499 synthetic construct for M.d. EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU500 synthetic construct for R.s. EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU503 synthetic construct for N.w. EG inserted into pMU451 (PacI/AscI digest of both pieces) pMU624/pMI529 2 micron vector for expression of T.e. CBH1 w/CBD (PCR fragments for chimeric enzyme with PmlI-XhoI digested pRDH105) pMU326 synthetic construct for R.s. EG from Codon Devices pMU784/pMI574 2 micron vector for expression of C.l.(b) CBH2 (synthetic construct for C.l.(b) CBH2 inserted into PacI/AscI digested pMU624) pMU562 pBKD_2 with loxp-zeo-loxp inserted (NotI digest of both pieces) pMU576 ENO1p- T.r.cbh1-ENO1.sub.T (from pMU291) in pMU562 (PacI/AscI digest of both pieces) pMU577 ENO1p- T.e.cbh1-ENO1.sub.T in (from pMU398) pMU562 (PacI/AscI digest of both pieces) pMU661 ENO1p- T.r. EG1-ENO1.sub.T (from pMU463) in pMU562 (PacI/AscI digest of both pieces) pMU662 ENO1p- C.l.(a) EG1-ENO1.sub.T (from pMU465) in pMU562 (PacI/AscI digest of both pieces) pMU663 ENO1p- C.f. EG1-ENO1.sub.T (from pMU471) in pMU562 (PacI/AscI digest of both pieces) pMU664 ENO1p- N.t. EG1-ENO1.sub.T (from pMU472) in pMU562 (PacI/AscI digest of both pieces) pMU665 ENO1p- C.a. EG1-ENO1.sub.T (from pMU473) in pMU562 (PacI/AscI digest of both pieces) pMU666 ENO1p- T.r.CBH2-ENO1.sub.T (from pMU475) in pMU562 (PacI/AscI digest of both pieces) pMU667 ENO1p- M.d.-EG1-ENO1.sub.T (from pMU499) in pMU562 (PacI/AscI digest of both pieces) pMU668 ENO1p- N.w.-EG1-ENO1.sub.T (from pMU503) in pMU562 (PacI/AscI digest of both pieces) pMU755 ENO1p- T.e.CBH1 w/CBD-ENO1.sub.T (from pMU624) in pMU562 (PacI/AscI digest of both pieces) pMU750 ENO1p- R.s.-EG2-ENO1.sub.T (from pMU326) in pMU562 (PacI/AscI digest of both pieces) pMU809 ENO1p- C.l.(b) CBH2b-ENO1.sub.T (from pMU784) in pMU562 (PacI/AscI digest of both pieces) pMU721 pMU562 with hph gene (hygromycin resistance marker) replacing zeocin marker (NotI digest for both fragments) pMU760 ENO1p- T.e.CBH1 w/CBD-ENO1.sub.T from pMU624 in pMU721 (MheI/AscI digest for both fragments) pMU761 ENO1p- T.r.CBH2-ENO1.sub.T from pMU291in pMU721 (PacI/AscI digest for both fragments) pMI553 2 micron vector for expression of T.r. CBH2 and T.e. CBH1 + CBM pMI568 2 micron vector for expression of T.r. EG1, please see text for description of how this construct was built. pMI574 2 micron vector for expression of C.l.(b) CBH2 pMI577 2 micron vector for expression of T.r. CBH2 and H.g. CBH1 pMI578 2 micron vector for expression of T.r. CBH2 and T.e. CBH1 pMI579 2 micron vector for expression of T.r. CBH2 and C.l.(b) CBH1 pMI580 2 micron vector for expression of C.l.(b) CBH2 and T.e. CBH1 + CBM pMI581 2 micron vector for expression of C.l.(b) CBH2 and T.e. CBH1 pMI582 2 micron vector for expression of C.l.(b) CBH2 and H.g. CBH1 pMI583 2 micron vector for expression of C.l.(b) CBH2 and C.t. CBH1

Abbreviations

[0258] ENO1.sub.P/T=Enolase 1 gene promoter/terminator, PGK1.sub.P/T=phosphoglycerate kinase 1 gene promoter & terminator; T.r.=Trichoderma reesei; H.g.=Humicola grisea; T.a.=Thermoascus aurantiacus; T.e.=Talaromyces emersonii, S.f.=Saccharomycopsis fibuligera; C.l. (a)=Coptotermes lacteus; C.f.=Coptotermes formosanus; N.t.=Nasutitermes takasagoensis; C.a.=Coptotermes acinaciformis; M.d.=Mastotermes darwinensis; N.w.=Nasutitermes walkeri; R.s.=Reticulitermes speratus; C.l. (b)=Chrysosporium lucknowense; N.f.=Neosartorya fischeri; R.f.=Reticulitermes flavipes; C.t.=Chaetomium thermophilum

TABLE-US-00008 TABLE 5 Primers Used sCBH1/2-L GACTGAATTCATAATGGTCTCCTTCACCTCC (SEQ ID NO: 59) sCBH1-R GACTCTCGAGTTACAAACATTGAGAGTAGTA TGG (SEQ ID NO: 60) sCBH2-R CAGTCTCGAGTTACAAGAAAGATGGGTTAGC (SEQ ID NO: 61) 395 Te cbh1 GCGTTGGTACCGTTTAAACGGGGCCCTTAAT Synt1 PacI-ATG TAAACAATGCTAAGAAGAGCTTTACTATTGA G (SEQ ID NO: 62) 398 Te cbh1 CCTCCCCCGGGTTAGAAGCAGTGAAAGTGGA synt core SmaI GTTGATTGG (SEQ ID NO: 63) 399Trcbh1 synt GCGACGAGFCAACCCICCAGGTOGTAACAGA CBM5 MlyIHincHII GGTACCAC (SEQ ID NO: 64) 400 Trcbh1 synt GCGACTCGAGGGCGCGCCTACAAACATTGAG CBM AscIXhoI AGTAGTATGGGTTTA (SEQID NO: 65) 379 ScPGK1prom- GCGTTGAGCTCGGGCCCTAATTTTTATTTTA 786 SacI + ApaI GATTCCTGACTTCAAC (SEQ ID NO: 66) 380 ScPGK1prom GCGTTGAATTCTTAATTAAGTAAAAAGTAGA EcoRI-PacI TAATTTACTCCTTG (SEQ ID NO: 67) 381 CBH2 WT GCGTTGAATTCTTAATTAAACAATGATTGTC EcoRI-PacI-ATG GGCATTCTCACCACGC (SEQ ID NO: 68) 386 CBH2 WTTAA- gcgatgaattcggcgcgccTTACAGGAACGA AscI-EcoRI TGGGTTTGCGTTTG (SEQ ID NO: 69)

[0259] The yeast expression vector YEpENO-BBH was created to facilitate heterologous expression under control of the S. cerevisiae enolase 1 (ENO1) gene promoter and terminator. The vector was also useful because the expression cassette from this vector could be simply excised using a BamHI, BglII digest. YEpENO1 (Den Haan et al., Metabolic Engineering. 9: 87-942007) contains the YEp352 backbone with the ENO1 gene promoter and terminator sequences cloned into the BamHI and HindIII sites. This plasmid was digested with BamHI and the overhang filled in with Klenow polymerase and dNTPs to remove the BamHI site. The plasmid was re-ligated to generate YEpENO-B. Using the same method, the BglII and then the HindIII sites were subsequently destroyed to create YEpENO-BBHtemplate. YEpENO-BBHtemplate was used as template for a PCR reaction with primers ENOBB-left (5'-GATCGGATCCCAATTAATGTGAGTTTACCTCA-3' (SEQ ID NO: 70)) and ENOBB-right (5'-GTACAAGCTTAGATCTCCTATGCGGTGTGAAATA-3' (SEQ ID NO: 71)) in which the ENO1 cassette was amplified together with a 150 bp flanking region upstream and 220 bp downstream. This product was digested with BamHI and HindIII and the over hangs filled in by treatment with Klenow polymerase and dNTPs and cloned between the two PvuII sites on yENO1 effectively replacing the original ENO1 cassette and generating YEpNO-BBH.

[0260] Codon optimized versions of Humicola grisea cbh1 (Hgcbh1), Thermoascus aurantiacus cbh1 (Tacbh1) and Talaromyces emersonii cbh1 and cbh2 (Tecbh1 and Tecbh2) were designed and synthetic genes were ordered from GenScript Corporation (Piscataway, N.J., USA). These four synthetic cbh encoding genes received from GenScript Corporation were cloned onto the plasmid pUC57. The resulting vectors were digested with EcoRI and XhoI to excise the cbh genes which were subsequently cloned into an EcoRI and XhoI digested YEpENO-BBH. This created the plasmids pRDH1003 (with Hgcbh1), pRDH104 (with Tacbh1), pRDH105 (with Tecbh1) and pRDH106 (with Tecbh2) with the cbh encoding genes under transcriptional control of the ENO1 promoter and terminator. Additionally, pRDH101 was created to express the T. reesei CBH1 from pBZD_10631_20641. Takara ExTaq enzyme was used as directed and to amplify the sTrcbh1 from pBZD_10631_20641 using primers sCBH1/2 L and sCBH1R. The fragment was then isolated and digested with EcoRI and XhoI. YEpENO-BBH was also digested with EcoRI and XhoI and the relevant bands were isolated and ligated. A 1494 bp fragment encoding the T. reesei cbh2 gene was amplified from the plasmid pBZD_10631_20641, with primers sCBH1/2-L and sCBH2 R (5'-CAGTCTCGAGTTACAAGAAAGATGGGTTAGC-3' (SEQ ID NO: 61)), digested with EcoRI and XhoI and cloned into the EcoRI and XhoI sites of pJC1 (Crouse et al., Curr. Gen. 28: 467-473 (1995)) placing it under transcriptional control of S. cerevisiae phosphoglycerate kinase 1 (PGK1) gene promoter and terminator. This plasmid was designated pRDH107. Subsequently the expression cassettes from pRDH103, pRDH104 and pRDH105 were excised with BamHI and BglII digestion and cloned into the BamHI site of pRDH107 to yield pRDH118, pRDH120, pRDH108 and pRDH109, respectively. pRDH109 contains the same expression cassettes as pRDH108 but in pRDH108 the gene expression cassettes are in the reverse orientation relative to each other. These plasmids and their basic genotypes are summarized in Table 4.

[0261] Two additional 2-micron vectors for expression of Chrysosporium lucknowense CBH2b and the T. emersonii CBH1 with a c-terminal fusion of the CBM of T. reesei CBH1 were also created. The fusion between T. emersonii cbh1 and the CBM of T. reesei cbh1 was generated by ligation of three fragments. Table 5 lists the oligonucleotides used for these constructs. A PCR product was amplified with the oligonucleotides 395 Te cbh1 Synt1 PacI-ATG and 398 Te cbh1 synt core SmaI using pRDH105 as the template, digested with PmlI and SmaI and the 800 bp fragment was isolated. A second PCR product was amplified with oligonucleotides 399 Trcbh1 synt CBM5 MlyHincII and 400 Trcbh1 synt CBM AscIXhoI with pRDH101 as the template, digested with MlyI and XhoI and the 180 bp fragment was isolated. The two PCR fragments were ligated with the 6.9 kb PmlI-XhoI fragment of pRDH105 resulting in pMU624.

[0262] The genomic 3900 bp DNA sequence of Chrysosporium lucknowense cbh2b gene (described in Published United States Patent Application No: 2007/0238155) was analyzed for putative introns using the NetAspGene 1.0 Server (http://www.cbs.dtu.dk/services/NetAspGene/). Removal of the predicted introns from the genomic sequence resulted in an open reading frame of 482 amino acids which was synthesized at Codon Devices and codon optimized for expression in S. cerevisiae and cloned into pUC57 vector. Plasmid pAJ401 (Saloheimo et al. Mol. Microbiol. 13:219-228, 1994), which contains the PGK1 promoter and terminator, was modified for expression of T. reesei cbh2 between PacI and AscI restrictions sites. The PGK1 promoter was amplified with primers 379 ScPGK1prom-786 SacI+ApaI and 380 ScPGK1prom EcoRI-PacI and pAJ410 as the template and digested with PacI and EcoRI. The T. reesei cbh2 ORF was amplified from pTTc01 (Teeri et al., Gene 51:43-52, 1987) with oligonucleotides 381 CBH2 WT EcoRI-PacI-ATG and 386 CBH2 WT TAA-AscI-EcoRI, digested with PacI and EcoRI, and ligated with the SacI-EcoRI digested pAJ401 resulting in pMI508. The PacI-AscI fragment in pMI508 was replaced by a synthetic 1.4 kb T. reesei egl1 gene resulting in pMI522. The 1.9 kb fragment of pMI522 was digested with PmlI and XhoI and ligated to the 6.4 kb PmlI-XhoI fragment of pRDH107 resulting in pMI568. pMI568 was digested with PacI and AscI and the 7 kb fragment was ligated to the 1.5 kb fragment of pMI558 producing pMU784 for the expression of C. lucknowense cbh2b.

[0263] A set of 2-micron vectors was also constructed for the expression of endoglucanases in S. cerevisiae, as well as related plasmids to act as controls. pMU451 was created as a control vector and for cloning the cellulases under control of the ENO1 promoter and terminator. This was done by adding a PacI/AscI linker into the EcoRI/XhoI site of pMU451. Synthetic genes ordered from Codon Devices and received in pUC57 were cloned into this vector as PacI/AscI fragments. Vectors created this way and listed in Table 4 are: pMU458, pMU463, pMU1465, pMU469, pMU471, pMU472, pMU1473, pMU475, pMU1499, pMU1500, and pMU503.

[0264] Vectors for integrating secreted versions of cellulases at the delta integration sites in S. cerevisiae, or for integration into the genome of K. marxianus were created from the pBKD_1 and pBKD_2 constructs. The S. fibuligera BGL1 (SfBGL1) was cloned by PCR from ySFI (van Rooyen et al., J. Biotechnol. 120: 284-95 (2005)). The endoglucanase (TrEGI) used was the sequence give in Table 1. The cellulase encoding genes were cloned via PCR (using PacI and AscI sites) into pBKD_1 and pBKD_2--to create pBKD1-BGL1 and pBKD2-sEG1. The ENO1P-sEG1-ENO1T cassette from pBKD2-sEG1 was subsequently sub cloned as a SpeI, NotI fragment to pBKD1-BGL1 to create pBKD1-BGL1-sEG1.

[0265] pMU562, used for integrating cellulases into K. marxianus, was generated by cutting with pMU185 (pUG66) with Not1 and isolating a 1190 bp lox P ZeoR containing insert. This insert was ligated into a Not1 digested 4.5Kb delta-integration vector to produce pMU562. pMU1576 was generated by cutting T. reesei CBH2 containing plasmid pMU291 with Asc1/Pac1, isolating a 1491 bp CBH2 gene and ligating it into delta-integration vector pMU562 cut with Asc1/Pac1. pMU577 was generated by cutting T. emersonii CBH1 from pMU398 with Asc1/Pac1, isolating a 1380 bp CBH1 gene and ligating into delta-integration vector pMU562 cut with AscI/PacI. Similarly, a set of recombinant cellulase constructs (pMI661 to pMU668 and pMU750, pMU755, pMU809--see Table 4), including a variety of endoglucanases and cellobiohydrolases, was incorporated into pMU562 for co-transformation. Synthetic sequences for these cellulase genes were originally obtained from Codon Devices and subsequently cloned into 2.mu. expression vectors for use in S. cerevisiae. They were then transferred from these vectors to the integrating vectors as detailed (including digests used) in Table X. Together these constructs formed a library that could be transformed separately or together and then screened by activity assay. Constructs were digested with enzymes that cut inside of, or very closely outside of, the delta sequences for integration. Similar constructs for integrating cellulases using the hygromycin marker (pMU721, pMU760, and pMU761) were also built.

[0266] Yeast Transformation

[0267] For routine transformation of whole plasmids in S. cerevisiae, standard chemical transformation was used (Sambrook et al. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press (1989)). For some transformations, a modified protocol described by Hill et al. (Nucleic Acids Res. 19: 5791 (1991)) was used.

[0268] A protocol for electrotransformation of yeast was developed based on Cho et al. (1999) and on Ausubel et al. (1994). Linear fragments of DNA were created by digesting pBD1-BGL1-sEG1 with AccI. AccI has a unique site in the .delta. sequence. The fragments were purified by precipitation with 3M NaAc and ice cold ethanol, subsequent washing with 70% ethanol, and resuspension in USB dH2O (DNAse and RNAse free, sterile water) after drying in a 70.degree. C. vacuum oven.

[0269] S. cerevisiae cells for transformation were prepared by growing to saturation in 5 mL YPD cultures. 4 mL of the culture was sampled, washed 2.times. with cold distilled water, and resuspended in 640 .mu.L cold distilled water. 80 .mu.L of 100 mM Tris-HCl, 10 mM EDTA, pH 7.5 (10.times.TE buffer--filter sterilized) and 80 .mu.L of 1 M lithium acetate, pH 7.5 (10.times.LiAc--filter sterilized) were added, and the cell suspension was incubated at 30.degree. C. for 45 min. with gentle shaking. 20 .mu.L of 1M DTT was added and incubation continued for 15 min. The cells were then centrifuged, washed once with cold distilled water, and once with electroporation buffer (1M sorbitol, 20 mM HEPES), and finally resuspended in 267 .mu.L electroporation buffer. The same protocol was used for transforming K. lactis and K. marxianus strains, except that 50 mLs of YPD was inoculated with 0.5 mL from an overnight culture, grown for 4 hours at 37.degree. C., and then centrifuged and prepared as above. Additionally, incubations and recovery steps were carried out at 37.degree. C.

[0270] For electroporation, 10 .mu.g of linearized DNA (measured by estimation on a gel) was combined with 50 .mu.L of the cell suspension in a sterile 1.5 mL microcentrifuge tube. The mixture was then transferred to a 0.2 cm electroporation cuvette, and a pulse of 1.4 kV (200.mu., 25 .mu.F) was applied to the sample using the Biorad Gene Pulser device. 1 mL of YPD with 1M sorbitol adjusted to pH 7.0 (YPDS) was placed in the cuvette and the cells were allowed to recover for .about.3 hrs. 100-200 .mu.L cell suspension were spread out on YPDS agar plates with appropriate antibiotic, which were incubated at 30.degree. (for 3-4 days until colonies appeared.

[0271] Yeast Strains

[0272] The yeast strains listed in Table 6 were created using the vectors and transformation protocols as described.

TABLE-US-00009 TABLE 6 Yeast Strains. Background Genes expressed and/or Name strain knocked out Constructs M0013 Saccharomyces Genotype: .alpha., leu2-3,112 ura3-52 None cerevisiae his3 trp1-289 Y294 (ATCC 201160) M0243 M0013 SfBGLI, TrEGI pBKD1-BGLI-sEGI M0244 M0013 SfBGLI, TrEGI (native sequence) pCEL5 M0247 M0013 TeCBH1; delta FUR1 pRDH105 M0248 M0013 TrCBH2, TeCBH1; delta FUR1 pRDH108; pDF1 M0249 M0013 None (control); delta FUR1 pJC1; pDF1 M0265 M0013 HgCBHI; delta FUR1 pRDH103; pDF1 M0266 M0013 TaCBHI; delta FUR1 pRDH104; pDF1 M0282 M0248 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; TeCBH1; delta FUR1 pRDH108; pDF1 M0284 M0243 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; HgCBH1; delta FUR1 pRDH118; pDF1 M0286 M0243 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; TaCBH1; delta FUR1 pRDH120; pDF1 M0288 M0243 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; TeCBH1; delta FUR1 pRDH108; pDF1 M0289 M0013 TrCBH2, HgCBH1; delta FUR1 pRDH118; pDF1 M0291 M0013 TrCBH2, TaCBH1; delta FUR1 pRDH120; pDF1 M0358 M0282 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; TeCBH1; delta FUR1; Trp1; His3 pRDH108; pDF1 M0359 M0288 SfBGLI, TrEGI, TrCBH2, pBKD1-BGLI-sEGI; TeCBH1; delta FUR1; Trp1; His3 pRDH108; pDF1 M0361 M0249 None (control); delta FUR1; pJC1; pDF1 Trp1; His3 M0157 Kluyveromyces None None marxianus (ATCC #10606) M0158 Kluyveromyces None None lactis (ATCC #34440) M0411 M0158 (colony SfBGLI, TrEGI pBKD1-BGLI-sEGI; #1) M0412 M0158 (colony SfBGLI, TrEGI pBKD1-BGLI-sEGI; #2) M0413 M0157 (Colony SfBGLI, TrEGI pBKD1-BGLI-sEGI; #1) M0414 M0157 (Colony SfBGLI, TrEGI pBKD1-BGLI-sEGI; #2) M0491 M0414 SfBGLI, TrEGI, TeCBH1, pBKD1-BGLI-sEGI; TrCBH2 pMU576 and pMU577 M0599 M0414 SfBGLI, TrEGI, TeCBH1, pBKD1-BGLI-sEGI; TrCBH2 pMU760 and pMU761 M0600 M0414 SfBGLI, TrEGI, TeCBH1, pBKD1-BGLI-sEGI; TrCBH2 pMU760 and pMU761 M0601 to M0414 (11 SfBGLI, TrEGI, pBKD1-BGLI-sEGI; M0604; colonies Cl(a)EG, CfEG, NtEG, CaEG, pMU663, pMU755, M0611 to displaying MdEG, NwEG, RsEG, TeCBH1, pMU809, pMU576, M0617 highest TeCBH1 + CBD, TrCBH2, pMU661, pMU662, avicelase Cl(b)CBH2 pMU664, pMU665, activity) pMU667, pMU668, pMU750, pMU577 M0618 to M0157(8 Cl(a)EG, CfEG, NtEG, CaEG, pMU663, pMU755, M0625 colonies MdEG, NwEG, RsEG, TeCBH1, pMU809, pMU576, displaying TeCBH1 + CBD, TrCBH2, pMU661, pMU662, highest Cl(b)CBH2 pMU664, pMU665, avicelase pMU667, pMU668, activity) pMU750, pMU577 yENO1 M0013 ENO1 P/T YEpENO-BBH; pDF1 M0419 M0013 ENO1 P/T pMU451 M0420 M0013 TeCBH1 pMU272 M0423 M0013 TrEG1 pMU463 M0424 M0013 SfBGL1 pMU464 M0426 M0013 RfEG pMU469 M0446 M0013 Cl(a)EG pMU465 M0449 M0013 CfEG pMU471 M0450 M0013 NtEG pMU472 M0460 M0013 MdEG pMU499 M0461 M0013 RsEG pMU500 M0464 M0013 NwEG pMU503 M0476 M0013 NfEG pMU458 Y294/pMI529 M0013 TeCBH1 + CBM pMU624 fur1.DELTA. Y294/pMI553 M0013 TrCBH2, TeCBH1 + CBM pMI553 fur1.DELTA. Y294/pMI574 M0013 Cl(b)CBH2 pMI574 fur1.DELTA. Y294/pMI577 M0013 TrCBH2, HgCBH1 pMI577 fur1.DELTA. Y294/pMI578 M0013 TrCBH2, TeCBH1 pMI578 fur1.DELTA. Y294/pMI579 M0013 TrCBH2, Cl(b)CBH1 pMI579 fur1.DELTA. Y294/pMI580 M0013 Cl(b)CBH2, TeCBH1 + CBM pMI580 fur1.DELTA. Y294/pMI581 M0013 Cl(b)CBH2, TeCBH1 pMI581 fur1.DELTA. Y294/pMI582 M0013 Cl(b)CBH2, HgCBH1 pMI582 fur1.DELTA. Y294/pMI583 M0013 Cl(b)CBH2, Cl(b)CBH1 pMI583 fur1.DELTA.

[0273] The plasmid pBKD1-BGL1-sEG1 (pMU276) was digested with AccI and transformed to S. cerevisiae Y294 by electrotransformation to create a strain with delta integrated copies of the SfBGL1 and TrEGI, designated M0243. Episomal plasmids were then transformed to S. cerevisiae Y294 and/or M0243.

[0274] To create autoselective S. cerevisiae strains, i.e. strains that can be grown in medium without requiring selective pressure to maintain the episomal plasmid, strains were transformed with NsiI & NcoI digested pDF1 and selected on SC-ura-leu plates. This lead to the disruption of the FUR1 gene of S. cerevisiae. PCR was used to confirm FUR1 disruption with primers FUR1-left (5'-ATTTCTTCTTGAACAATGAAC-3' (SEQ ID NO: 72)) and FUR1-right (5'-CTTAATCAAGACTTCTGTAGCC-3' (SEQ ID NO: 73)), where a 2568 bp indicated a disruption.

[0275] M0282 was created by transforming M0248 with AccI digested pBKD1-BGL1-sEGI, as described above, except that the transformation mixture was spread on plated containing 10 g/L BMCC with 10 g/L yeast extract and 20 g/L peptone.

[0276] The presence of integrated genes was verified by colony PCR for Kluyveromyces strains. Selected yeast strains were made prototrophic by transforming with PCR products for genes to complement their auxotrophies.

[0277] Cellulosic Substrates for Enzyme Assays

[0278] Bacterial microcrystalline cellulose (BMCC) was a gift from CP Kelco company. BMCC as received was stirred O/N at 4 C in water. After the substrate was rehydrated, it was washed 6 times with water and resuspended in water. The dry weight of the substrate was measured by drying samples at 105 C until constant weight was obtained.

[0279] Avicel PH105 (FMC Biopolymers) was used as provided by the manufacturer.

[0280] Pretreated mixed hardwoods were generated by autohydrolysis of the substrate at 160 PSI for 10 minutes. Pretreated material was washed 5 times to remove inhibitors and soluble sugars and resuspended in distilled water. Samples were dried overnight at 105 C to determine the dry weight. Analysis of sugar content by quantitative saccharification showed a 50% glucan content.

[0281] Phosphoric acid swollen cellulose (PASC) was prepared as in Zhang and Lynd (2006), with only slight modifications. Avicel PH105 (10 g) was wetted with 100 mL of distilled water in a 4 L flask. 800 mL of 86.2% phosphoric acid was added slowly to the flask with a first addition of 300 mL followed by mixing and subsequent additions of 50 mL aliquots. The transparent solution was kept at 4.degree. C. for 1 hour to allow complete solubilization of the cellulose, until no lumps remained in the reaction mixture. Next, 2 L of ice-cooled distilled water was added in 500 mL aliquots with mixing between additions. 300 mL aliquots of the mixture were centrifuged at 5,000 rpm for 20 minutes at 2.degree. C. and the supernatant removed. Addition of 300 mL cold distilled water and subsequent centrifugation was repeated 4.times.. 4.2 mL of 2M sodium carbonate and 300 mL of water were added to the cellulose, followed by 2 or 3 washes with distilled water, until the final pH was .about.6. Samples were dried to constant weight in a 105.degree. C. oven to measure the dry weight.

[0282] Enzyme Assays

[0283] .beta.-glucosidase activity was measured in a manner similar to McBride, J. E., et al., (Enzyme Microb. Techol. 37: 93-101 (2005)), except that the volume of the assay was decreased and the reaction performed in a microtiter plate. Briefly, yeast strains were grown to saturation in YPD or YPC media with or without appropriate antibiotics, the optical density at 600 nm (OD(600)) was measured, and an 0.5 mL sample of the cultures was taken. This sample was centrifuged, the supernatant was separated and saved, and the cell pellet was washed 2.times.50 mM citrate buffer, pH 5.0. Reactions for supernatants were made up of 50 .mu.L sample, 50 .mu.L citrate buffer, and 50 .mu.L 20 mM p-nitrophenyl-.beta.-D-glucopyranoside (PNPG) substrate. Reactions with washed cells consisted of 25 .mu.L of cells, 75 .mu.L citrate buffer, and 50 .mu.L PNPG substrate. If the activity was too high for the range of the standard curve, a lower cell concentration was used and the assay was re-run. The standard curve consisted of a 2-fold dilution series of nitrophenol (PNP) standards, starting at 500 nM, and ending at 7.8 nM, and a buffer blank was included. After appropriate dilutions of supernatant or cells were prepared, the microtiter plate was incubated at 37.degree. C. for 10 minutes along with the reaction substrate. The reaction was carried out by adding the substrate, incubating for 30 min., and stopping with 150 .mu.L of 2M Na.sub.2CO.sub.3. The plate was then centrifuged at 2500 rpm for 5 minutes, and 150 .mu.L of supernatant was transferred to another plate. The absorbance at 405 nm was read for each well.

[0284] Endoglucanase activity was qualitatively detected by observing clearing zones on synthetic complete media (as above, but including 20 g/L glucose) plates with 0.1% carboxymethyl cellulose (CMC) stained with congo red (Beguin, Anal. Biochem. 131: 333-6 (1983)). Cells were grown for 2-3 days on the plates and were washed off the plate with 1M Tris-HCL buffer pH 7.5. The plates were then stained for 10 minutes with a 0.1% Congo red solution, and extra dye was subsequently washed off with 1M NaCl.

[0285] CBH1 activity was detected using the substrate 4-Methylumbelliferyl-.beta.-D-lactoside (MULac). Assays were carried out by mixing 50 .mu.L of yeast supernatant with 50 .mu.L of a 4 mM MUlac substrate solution made in 50 mM citrate buffer pH 5.5. The reaction was allowed to proceed for 30 minutes and then stopped with 1M Na.sub.2CO.sub.3. The fluorescence in each well was read in a microtiter plate reader (ex. 355 nm and em. 460 nm).

[0286] Quantification of Enzyme Activity

[0287] Enzyme activity on PASC and Avicel were measured using the protocol described in Den Haan et al., Enzyme and Microbial Technology 40: 1291-1299 (2007). Briefly, yeast supernatants were incubated with cellulose at 4.degree. C. to bind the cellulase. The cellulose was then filtered from the yeast supernatant, resuspended in citrate buffer and sodium azide, and incubated at 37.degree. C. Accumulation of sugar was measured in the reaction by sampling and performing a phenol-sulfuric acid assay. (See Example 10 and Table 9.)

[0288] Avicel activity levels were also generated using a 96-well plate method. (See Example 2.) Strains to be tested were grown in YPD in deep-well 96 well plates at 35.degree. C. with shaking at 900 RPM. After growing, plates were centrifuged at 4000 rpm for 10 min. 300 .mu.L substrate (2% avicel, 50 mM sodium acetate buffer, 0.02% sodium azide, .beta.-glucosidase--1 .mu.L per mL) was added to a new 96-well deep well plate, without allowing the avicel to settle. 300 .mu.L of yeast supernatant was added to this substrate, and 100 .mu.L was taken for an initial sample. The assay plate is incubated at 35.degree. C., with shaking at 800 rpm, and samples were taken at 24 and 48 hours. Samples were placed in 96-well PCR plates, and spun at 2000 rpm for 2 minutes. 50 .mu.L of supernatant was then added to 100 .mu.L of DNS reagent previously placed in a separate 96 well PCR plate, mixed, and heated to 99.degree. C. for 5 minutes in a PCR machine, followed by cooling to 4.degree. C. 50 .mu.L was transferred to a microtiter plate and the absorbance was measured at 565 nm. The conversion of avicel was calculated as follows:

Y = ( OD ( T = 24 or 48 ) - OD ( T = 0 ) ) .times. 100 % = .DELTA. OD .times. 100 S .times. A = .DELTA. OD .times. 100 0.1 .times. 10 ##EQU00001##

Y--% of Avicel converted at 24 or 48 hrs S--DNS/glucose calibration slope that is 0.1 for DNS at 565 nm A--Avicel concentration at T=0 that is 10 g/L for 1% Avicel

Example 1: Production of Kluyveromyces Expressing Heterologous .beta.-Glucosidase and Endoglucanase

[0289] In order to test the ability of Kluyveromyces to express functional heterologous cellulases, two Kluyveromyces strains, Kluyveromyces marxianus (ATCC strain #10606; MO157) and Kluyveromyces lactis (ATCC strain #34440), were transformed with vectors encoding heterologous cellulases.

[0290] Vectors containing yeast delta integration sequences, the KanMX marker and sequences encoding S.f. BGL1 and T.r. EGI (pBKD-BFLI-sEG1) were transformed into Kluyveromyces according to the yeast transformation protocol as described above, and selected on G418. Transformants were verified by PCR and then tested by CMC assay. The results are shown in FIG. 1. The presence of the heterologous cellulase activity is indicated by a clearing zone on the CMC plate. As shown in FIG. 1, neither an untransformed K. lactis strain (colony 8) or an untransformed K. marxianus strain (colony 16) showed endoglucanase activity. However, 6 of 7 transformed K. lactis colonies showed CMCase activity, and all 7 transformed K. marxianus colonies showed CMCase activity. MO413 and MO414 were identified as two K. marxianus colonies showing CMCase activity.

Example 2: Production of Kluyveromyces Expressing CBH1 and CBH2

[0291] The ability of Kluyveromyces to express functional heterologous cellobiohydrolases was also examined. In these experiments, K. marxianus (MO157) was transformed with constructs containing T. reesei CBH2, T. emersonii CBH1 or both. Similarly, MO414 (K. marxianus transformed with S.f. BGLI and T.r. EGI) was transformed with constructs containing T. reesei CBH2, T. emersonii CBH1 or both.

[0292] Transformations were performed as described in above. CBH1 activity was then detected using the substrate 4-Methylumbelliferyl-.beta.-D-lactoside (MU-Lac) as described above. The assay was performed on eight colonies of each transformant and the three colonies showing the highest activity were averaged. The results are shown in FIG. 2 and demonstrate that strains transformed with T. emersonii CBH1 had high MU-lac activity.

[0293] The activity of Kluyveromyces strains expressing heterologous cellobiohydrolases on Avicel was also assessed. In one experiment, MO413 was transformed with vectors containing T. reesei CBH2 and T. emersonii CBH1 coding sequences along with a zeocin marker. Novel strain MO491 was created by this transformation and showed MU-lactoside activity. In a second experiment, MO413 was transformed with vectors containing T. reesei CBH2 and T. emersonii CBH1 coding sequences along with a hygromycin marker, and strains MO599 and MO600 were isolated from this transformation. Activity on Avicel was assessed at 48 hours as described above, and the results, shown in FIG. 3, demonstrate that Kluveryomyces expressing heterologous cellulases have Avicelase activity at 35.degree. C. Avicelase activity at 45.degree. C. was also demonstrated (data not shown).

Example 3: Production of Kluyveromyces Expressing a Library of Cellulases

[0294] Kluyveromyces strains were also created by transforming yeast with a library of cellulases (creation of library was described above). For example, MO413 was transformed with a library of cellulases containing a zeocin marker to produce novel strains MO601-MO604 and MO611-MO617. In addition, MO157 (K. marxianus) was transformed with the same library and novel strains MO618-MO625 were identified. Activity on Avicel was assessed at 48 hours as described above, and the results, shown in FIG. 3, demonstrate that Kluveryomyces transformed with a library of heterologous cellulases also have Avicelase activity at 35.degree. C. Transformants of M0157 with the library showed the highest activity. Avicelase activity at 45.degree. C. was also demonstrated (data not shown).

Example 4: Ethanol Production by Transformed Kluyveromyces

[0295] In order to determine if Kluyveromyces expressing heterologous cellulases could produce ethanol from Avicel, precultures were grown in for 24 hours in YPD (YPD as above, with 20 g/L glucose; 25 mL, in a 250 mL shake flask) with shaking at 300 rpm at 35.degree. C. After 24 and 48 hours, 40 g/L of additional glucose was added. At 72 hours, the pH of the cultures was adjusted to .about.5.0 with citrate buffer (initial pH of buffer was 5.5, final concentration was 50 mM), and the culture was added to a sealed plastic shake flask containing 5.5 grams of Avicel (final concentration 10% (w/v). Avicel PH105 (FMC Biopolymers) was used as provided by the manufacturer. The culture was incubated at 35.degree. C. with shaking at 150 rpm.

[0296] Quantification of ethanol in fermentation samples was carried out by HPLC analysis, and initial ethanol concentrations in bottles (from precultures) was subtracted from all subsequent data points (initial ethanol concentrations ranged between 0 and about 6 g/L). The initial glucose concentration for all strains except MO603 was 0.000 g/L. For this strain it was 0.069 g/L, which would result in a maximum in 0.035 g/L of ethanol from the initial sugar.

[0297] The results, as shown in FIG. 4, demonstrate that Engineered K. marxianus strains were also able to produce ethanol directly from Avicel. Strain MO157, the untransformed control, showed a steady decrease in ethanol concentration over the course of the experiment. This is clue to ethanol consumption by the strain because of the presence of a small amount of oxygen in the flasks.

[0298] Of the two strains transformed with T. reesei CBH2 and T. emersonii CBH1 with the hygromycin marker (MO599 and M600), one (MO599) showed ethanol production. In addition, of the five strains transformed with T. reesei CBH2 and T. emersonii CBH1 with the zeocin marker, four (MO601, MO602, MO604 and MO491) showed ethanol production. This demonstrates that engineered thermotolerant K. marxianus are capable of producing ethanol directly from the recalcitrant crystalline cellulose, Avicel.

Example 5: Production of S. cerevisiae Expressing Heterologous Cellulases

[0299] S. cerevisiae expressing heterologous cellulases were also produced and tested for their ability to grow on media containing bacterial microcrystalline cellulose (BMCC). In these experiments, microaerobic conditions were maintained by growing strains on BMCC in sealed hungate tubes with an air atmosphere.

[0300] Strains expressing T. emersonii CBH1 and T. reesei (CBH2 (MO248) were transformed with a construct allowing T. reesei GI and S. fibuligera BGLI expression (pKD-BGLI-sEGI). That transformation was plated on a BMCC solid agar plate and five colonies appeared on the plate after seven days (data not shown). Yeast from the largest of the five colonies was isolated as strain MO282. (MO282 is described in more detail above.) The three control strains were tested for growth on the same plates. One strain expressed with T. emersonii CBH1 and T. reesei CBH2, and two strains expressed T. reesei EGI and S. fibuligera BGLI. No colonies appeared on plates with control yeast strains (data not shown).

[0301] The ability of MO282 to grow on BMCC was also tested using liquid media. FIG. 5 shows that MO282, which expresses all 4 secreted cellulases grew to a much greater extent on BMCC than a plasmid only control (MO249), a strain expressing only T. emersonii CBH1 and T. reesei CBH2 (MO249), and a strain expressing 4 tethered cellulases (MO144).

[0302] These results indicate that yeast expressing secreted T. emersonii CBH1, T. reesei CBH2, T. reesei EGI and S. fibuligera BGLI heterologously are able to grow on bacterial microcrystalline cellulose.

Example 6: S. cerevisiae Expressing Heterologous Cellulases can Produce Ethanol from Avicel and Pretreated Hardwood

[0303] In order to determine if transformed S. cerevisiae can produce ethanol directly from cellulose without exogenously added cellulase enzymes, transformed strains were grown on Avicel as the sole carbon source. Avicel PH105 (FMC Biopolymers) was used as provided by the manufacturer.

[0304] Avicel media was made using the non-glucose components of synthetic complete medium for yeast including, yeast nitrogen base without amino acids--6.7 g/L, and supplemented with a complete amino acid mix (complete supplemental mixture). In some cases yeast extract (10 g/L) and peptone (20 g/L) (YP) were used as supplements in growth experiments. Cultivation conditions were anaerobic and were maintained by flushing scaled glass bottles with N2 after carbon source addition and before autoclaving. Non-carbon media components were added as 10.times. solutions by filter sterilizing after autoclaving. Inoculation into Avicel cultures was done at 20% by volume. Quantification of ethanol in fermentation samples was carried out by HPLC analysis, and initial ethanol concentrations in bottles (from precultures) was subtracted from all subsequent data points.

[0305] As shown in FIG. 6, Strain M0288 (expressing S. fibuligera BGLI, T. reesei EGI, T. reesei CBH2, and T. emersonii CBH1) was able to produce ethanol directly from avicel PH105 as compared to the control strain (M0249) when YNB media components were used.

[0306] The ability of MO288 to produce ethanol from cellulose was also demonstrated using pretreated hardwoods. Pretreated mixed hardwoods were generated by autohydrolysis of the substrate at 160 PSI for 10 minutes. Pretreated material was washed 5 times to remove inhibitors and soluble sugars and resuspended in distilled water. Samples were dried overnight at 105.degree. C. to determine the dry weight. Analysis of sugar content by quantitative saccharification showed a 50% glucan content. Media and culture conditions were as described above for Avicel experiments except that cultures were inoculated at 10% by volume.

[0307] The data presented in FIG. 7 demonstrates that MO288 was also able to make ethanol from pretreated hardwoods without added enzyme. The strain made .about.0.5 g/L more than the control when YP was used as media, and .about.0.2 g/L when YNB was used.

[0308] These data demonstrate that yeast expressing secreted T. emersonii CBH1, T. reesei CBH2, T. reesei EGI and S. fibuligera BGLI heterologously are able to produce ethanol from cellulose without the addition of any exogenous cellulases.

Example 7: Transformed Yeast Strains and Externally Added Cellulases Act Synergistically to Produce Ethanol from Pretreated Mixed Hardwoods

[0309] Production of ethanol from biomass is currently achieved using an SSF type of process where cellulase enzymes are added exogenously to a reaction containing pretreated cellulosic biomass, yeast growth media, and yeast. In order to determine if yeast expressing recombinant cellulases could improve this process, recombinant yeast expressing secreted cellulases were cultured in the presence of a range of exogenously added cellulase concentrations. Growth and media conditions were as described in previous examples.

[0310] In these experiments, a recombinant yeast strain expressing four secreted cellulases (MO288) was compared directly to the control strain (MO249) under the same conditions. External cellulases were added at concentrations of 25 mg cellulase per gram cellulose (100%), 22.5 mg cellulase per gram cellulose (90%), 18.75 mg cellulase per gram cellulose (75%) or 6.25 mg cellulase per gram cellulose (25%). Experiments were also performed without adding any external cellulases (0%). Pretreated mixed hardwoods (prepared as described in examples above) at an initial solids concentration of 5% were used as a cellulose source. The data is presented in FIG. 8. From this data, it is clear that the strain producing cellulases makes additional ethanol relative to the control strain for each of the cellulase loading concentrations tested.

[0311] In order to examine this effect in more detail, ethanol production at different external cellulase concentrations was evaluated in two different types of media using pretreated mixed hardwood. The results are shown in FIG. 9. In YP media, MO288 makes 6-9% more ethanol at the higher cellulase loadings, only 1% more at a 25% loading, and 100% more when no cellulase is loaded. In YNB media MO288 makes 20-40% more ethanol at low cellulase loadings, and .about.10% more ethanol at higher cellulase loadings. These results can be used to determine the amount of cellulase that can be removed from the process with the same overall ethanol yield being achieved. For YP media cellulase loading can be reduced .about.15% compared to the control, and for YNB media, cellulase loading can be reduced .about.5%. At non-zero cellulase loadings ethanol productivity was increased between 5 and 20% for strains expressing cellulases in YP media as compared to the control. It was increased between 10 and 20% for strains cultured in YNB media compared to the control.

[0312] These data demonstrate that previous SSF processes can be improved in terms of ethanol yield from biomass and ethanol productivity if strains expressing secreted cellulases are used in combination with exogenously added cellulases. Similarly, cellulase loadings required to achieve a particular percentage of theoretical ethanol yield can be reduced when strains expressing recombinant cellulases are added.

Example 8: Transformed Yeast Strains Also Increase Efficiency of Externally Added Cellulases in the Production of Ethanol from Avicel

[0313] To test whether this same trend would hold at high substrate concentrations these experiments were repeated using 15% Avicel PH1105 as substrate instead of 5% pretreated mixed hardwood. The results are shown in FIGS. 10 and 11. The strain making cellulases (MO288) routinely produced more ethanol from Avicel than the control yeast strain (MO249) under identical conditions, even at increased ethanol concentrations (FIG. 10). For example, when 25 mg cellulase per gram cellulose was loaded in the SSF reaction, the test strain (M0288) produced 54 g/L, while the control (M0249) produced 50 g/L.

[0314] To examine cellulase displacement the percentage of theoretical ethanol yield achieved at different cellulase loadings was determined. The results presented in FIG. 12 were repeated in triplicate for M0288 and M0249, allowing standard deviations for the increased ethanol yields to be calculated. The data that can be used for calculating cellulase displacement is presented in FIG. 12. FIG. 12 presents cellulase enzyme savings based on theoretical ethanol yield at 168 hours in an SSF experiment. SSF was performed in 30 ml of nitrogen purged YP+15% Avicel in pressure bottles. External cellulase mix at a ratio of 5 Spyzme:1 Novozyme-188 was used. The experiment was continued for 168 hours and sampling was done each day for ethanol estimation by HPLC. The arrows in the figure depict the necessary cellulase loading needed to achieve the same ethanol production from cellulose as the control. This loading is consistently lower than for the control (i.e. the ethanol yield is consistently higher). For data at 168 hours, the average cellulase displacement (amount less that needs to be loaded) is 13.3%.+-.4.9%.

Example 9: Use of Artificial Cbh1 to Produce Ethanol

[0315] In order to design a CBH1 protein with efficient cellulase activity, 17 CBH1 protein sequences from NCBI database (Table 7) were aligned.

TABLE-US-00010 TABLE 7 Fungal CBH1 genes used for alignment. Organism Genbank# Neosartorya fischeri XM_001258277 Gibberella zeae AY196784 Penicillium janthinellum X59054 Nectria haematococca AY502070 Fusarium poae AY706934 Chaetomium thermophilum AY861347 Aspergillus terreus XM_001214180 Penicillium chrysogenum AY790330 Neurospora crassa X77778 Trichoderma viride AY368686 Humicola grisea X17258 Thermoascus aurantiacus AF421954 Talaromyces emersonii AAL89553 Trichoderma reesei P62694 Phanerochaete chrysosporium Z29653 Aspergillus niger XM_001391971 Aspergillus niger XM_001389539

[0316] The artificial protein sequence was designed as a consensus (the most common) sequence for these proteins. The predicted signal sequence was exchanged by S. cerevisiae alpha mating factor pre signal sequence, and the sequence of the consensus CBH1 protein is shown below. Capital letters indicate the S. cerevisiae alpha mating factor pre signal sequence.

TABLE-US-00011 (SEQ ID NO: 43) MRFPSIFTAVLFAASSALAqqagtltaethpsltwqkctsggscttvngs vvidanwrwvhatsgstncytgntwdttlcpddvtcaqncaldgadysst ygvttsgnslrlnfvtqgsqknvgsrlylmeddttyqmfkllgqeftfdv dsnlpcglngalyfvamdadggmskypgnkagakygtgycdsqcprdlkf ingqanvegwepssndanagignhgsccaemdiweansistaftphpcdt igqtmcegdscggtyssdryggtcdpdgcdfnpyrmgnktfygpgktvdt tkkvtvvtqfitgssgtlseikrfyvqngkkvipnsestisgvsgnsitt dfctaqktafgdtddfakkgglegmgkalaqgmvlvmslwddhaanmlwl dstyptdatsstpgaargscdtssgvpadveanspnsyvtfsnikfgpig stftg.

[0317] An S. cerevisiae and K. lactis codon optimized sequence for expressing the CBH1 consensus sequence (SEQ ID NO:44) was developed and is shown below.

TABLE-US-00012 (SEQ ID NO: 44) atgagattccttcaatcttctgagttttgttcgcagcctcattgtgcttt attcacaacaggccggaacattgacattcagaaactcatccttccttaac ctggcaaaagtgcacttctgttaggttcatgcactacagtgaatggatct gtcgtgatcgatgcaaactggagatgggttcacgcaacttcaggttctac caactgttataccggaaacacttgggacaccacattgtgcccagatgacg tcacgtgcgctcagaactgtgctttggatggagctgattacagttcaacc tatggtgtaactacatccggaaactctttgagattaaacttcgttactca aggaagtcaaaagaacgttggttctagattgtacttaatggaggacgata caacctatcaaatgttcaaattgttaggtcaggagttcacctttgacgta gatgtcagtaacttgccatgtgggttaaacggagctttatactttgtggc aatggatgctgacggtggaatgtccaagtatccaggaaacaaagccggtg caaagtacggtacaggatattgtgattcacagtgccctagagatttgaag ttcattaacggtcaagcaaatgtggagggttgggaaccatctagtaacga tgccaatgcgggtattggtaatcatgggtcctgttgcgctgagatggata tctgggaggccaactcaatatctactgcctttacccctcacccatgcgat acaattggtcaaactatgtgcgagggtgattcatgtggtggaacctactc ctctgatacgatacggaggtacatgcgatccagatggttgcgactttatc catacagaatgggaaacaaaaccttttacggtcctggaaagacagttgat actaccaagaaagtaacagtcgtgacccagtttatcaccggtagttctgg aaccttatccgaaatcaaaagattctacgttcagaacggtaaagtaattc caaacagtgaatctacaatttcaggagtgagtggtaattctattactacc gacttttgtacagctcagaaaacagcatttggtgacaccgatgactttgc taagaagggtggattagaaggtatgggtaaagctttggcccagggaatgg tgttagttatgtctttatgggatgatcacgccgcaaatatgttatggttg gattcaacatatccaactgatgccacaagtagtacacctggagctgccag aggttcttgtgatacatcttccggtgttccagccgatgtagaagcaaatt ctcctaactcctatgttaccttctccaatataaagtttggtccaatcggt tcaacattcactggttaa

[0318] The codon optimized sequence was inserted into the episomal yeast expression vector (pMU451) under control of ENO1 promoter and terminator into PacI/AscI sites. The resulting expression constructs (pMU505) was transformed into M0375 host strain that derived from Y294 (MO013) in which His3 and Trp1 auxotrophies were rescued by transformation with S. cerevisiae His3 and Trp1 PCR products. The resulting strain expressing the CBH1 consensus sequence was named MO429.

[0319] In order to determine if MO429 had cellulase activity, an Avicel conversion assay was performed as described above and measured at 24 hours. As shown in FIG. 14, S. cerevisiae expressing the consensus Cbh1 sequence (MO429) showed cellulase activity as compared to a negative control transformed with an empty vector (MO419). The cellulase activity of MO429 was also compared to that of yeast strains expressing other heterologous cellulases. The strains tested are summarized in Table 8 below.

TABLE-US-00013 TABLE 8 Cellulytic Strains Used in Avicel Conversion Assay Strain # Description Cellulose Family Organism Activity Signal M0419 MO375 + pM none none U451 M0420 MO375 + pM CBH1 Fungi Talaromyces exo native U272 emersonii M0429 MO375 + pM fungal Fungi N/A exo S.c..alpha.MFpre U505 CBH1 consensus M0445 MO375 + pM CBH1 Fungi Neosartorya exo S.c..alpha.MFpre U459 fischeri M0456 MO375 + pM CBH1 Fungi Chaetomium exo S.c..alpha.MFpre U495 thermophilum M0457 MO375 + pM CBH1 Fungi Aspergillus exo S.c..alpha.MFpre U496 terreus M0458 MO375 + pM CBH1 Fungi Penicillium exo S.c..alpha.MFpre U497 chrysogenum

[0320] All of the strains in Table 8 were derived from the same parental MO375 strain and were transformed with an episomal yeast vector. MO420, MO429, MO445, MO456, MO457 and MO458 were created using episomal yeast vectors containing the heterologous cellulase genes as listed in the table which were codon optimized for expression in S. cerevisiae and K. lactis. The cellulases in MO429, MO445, MO456, MO457 and MO458 were expressed under control of S. cerevisiae ENO1 promoter and terminator. T. emersonii CBH1 was expressed with its own native signal sequence. As shown in FIG. 14, the secreted activity on Avicel of the consensus CBH1 was comparable with activity of other fungal CBH1s expressed in the same vector and in the same host strain.

Example 10: Comparison of Cellulase Activity in S. cerevisiae

[0321] S. cerevisiae were transformed with polynucleotides encoding a number of different heterologous cellobiohydrolases and their activity on PASC and Avicel was assessed as described above. The results are shown in the table below:

TABLE-US-00014 TABLE 9 Cellobiohydrolase activity in S. cerevisiae. Act. (PASC) Act. (Avicel) Plasmid Expression Cassette(s) (mU/gDCW) (mU/gDCW) yENO1 ENO1p/t 2.68 .+-. 1.1 2.99 .+-. 0.7 M0265 ENO1p/t-sH.g.cbh1 32.82 .+-. 6.5 34.85 .+-. 2.0 M0266 ENO1p/t-sT.a.cbh1 38.56 .+-. 5.9 38.15 .+-. 4.1 M0247 ENO1p/t-sT.e.cbh1 75.60 .+-. 13.1 21.42 .+-. 6.1 M0248 PGK1p/t-sT.r.cbh2 & 174.35 .+-. 6.5 40.5 .+-. 4.9 ENO1p/t-sT.e.cbh1 M0289 PGK1p/t-sT.r.cbh2 & Not measured 106.2 .+-. 6.8 ENO1p/t-sH.g.cbh1 M0291 PGK1p/t-sT.r.cbh2 & Not measured 32.7 .+-. 5.7 ENO1p/t-sT.a.cbh1

[0322] In addition, activity on Avicel was assayed using a 96-plate assay, and the results are shown in FIG. 14. In the Figure, for each strain, the first bar indicates the sugar released at 24 hours, and the second bar indicates the sugar released by 48 hours. CBH1s expressed individually, or in combination with T. reesei CBH2 showed some avicel activity--reaching 10% conversion of avicel in 48 hours. Combinations of CBH1 with CBH2 from C. lucknowense reached much higher avicel conversions of about 22% conversion in 48 hours in combination with T. emersonii CBH1 with CBD attached.

[0323] The avicel activity data for endoglucanases tested in S. cerevisiae is shown in FIG. 15. The data demonstrate that among the EGs tested the C. formosanus EG demonstrated the highest avicel activity when expressed in S. cerevisiae.

Example 11: Co-Cultures of Yeast Strains Expressing Different Heterologous Cellulases Produce Ethanol from Avicel

[0324] A co-culture of a number of cellulase producing yeast strains also showed the ability to make ethanol from Avicel PH105 in YNB media (FIG. 16). In this experiment 5 strains independently producing T. emersonii CBH1 (M0247), T. aurantiacus CBH1 (M0266), H. grisea CBH1 (M0265), a combination of T. emersonii CBH1 and T. reesei CBH2 (M0248), and a combination of T. reesei EGI and S. fibuligera BGL1 (M0244) were mixed in equal proportion by volume and then inoculated at 20% by volume. Each of the heterologously expressed cellulases in each of these strains was secreted. Media and culture conditions were as described above for Avicel experiments. The data in FIG. 16 demonstrate that heterologous cellulases do not need to be expressed in an individual yeast strain in order to produce ethanol from cellulose. Instead, yeast strains expressing different secreted heterologous cellulases can be cultured together in order to produce ethanol from cellulose without the addition of any exogenous cellulases.

[0325] A co-culture using a different combination of cellulases was also evaluated. In this set of co-culture experiments, four yeast strains were cultured together: M0566 (M0424 with FUR deletion): Secreted SfBGLI; M0592 (M0449 with FUR deletion): Secreted CfEGI; M0563 (same as Y294/pMI574 fur1.DELTA.): Secreted C1 CBH2b; and M0567 (same as Y294/pMI529 fur1.DELTA.): Secreted TeCBH1+CBD. These strains were grown in liquid YPD for 3 days, until the culture was saturate for pre-culture. At this point they were used to inoculate experiments where avicel (10%) was used as the substrate, and the 4 strains were mixed at equal volume prior to inoculation.

[0326] FIG. 17 demonstrates that the co-cultured strains are capable of producing ethanol directly from avicel in the absence of any added cellulase enzyme. The co-culture produces about 4-fold more ethanol after 168 hours as compared to the control strain, and about 3-fold more than M0288.

[0327] This co-culture was also used in SSF experiments where Zoomerase cellulase enzyme cocktail was used at 5 different loadings (10 mg protein/g avicel, 7.5 mg/g, 5 mg/g, and 2.5 mg/g, and 0 mg/g), and strains were inoculated at 10% by volume.

[0328] FIG. 18 presents the raw data for ethanol production at a variety of cellulase loadings by the co-culture, M0288, and M0249. FIG. 18A shows that at all cellulase loadings tested, the co-cultured strains produced significantly more ethanol than a control not producing cellulase. FIG. 18B shows that at all cellulase loadings tested, the co-culture produced more ethanol than the previously tested strain M0288. FIG. 19 shows the percentage of the theoretical yield of ethanol that could be achieved with each of these cultures after 168 hours of SSF using a variety of cellulase loadings. The data demonstrate that the co-cultured strains would achieve about a 2-fold reduction in cellulase relative to the control strain, and approximately a 35% reduction compared to M0288.

[0329] These data demonstrate that the combination of cellulases in this co-culture is highly efficient in the production of ethanol.

Example 12: Construction of a Robust Xylose-Utilizing Strain

[0330] M0509 (ATCC deposit designation ______, deposited on Nov. 23, 2009) is a strain of Saccharomyces cerevisiae that combines the ability to metabolize xylose with the robustness required to ferment sugars in the presence of pretreated hardwood inhibitors. M0509 was created in a three-step process. First, industrial strains of S. cerevisiae were benchmarked to identify strains possessing a level of robustness/hardiness sufficient for simultaneous saccharification and fermentation (SSF) of pretreated mixed hardwood substrates. Strain M0086, a diploid strain of strain of S. cerevisiae, satisfied this first requirement. Second, M0086 was genetically engineered with the ability to utilize xylose, resulting in strain M0407. Third, M0407 was adapted for several weeks in a chemostat containing xylose media with pretreatment inhibitors, generating strain M0509.

[0331] Strain M0407 was genetically engineered from M0086 to utilize xylose. This engineering required seven genetic modifications. The primary modification was the functional expression of the heterologous xylose isomerase gene, XylA, isolated from the anaerobic fungus Piromyces sp. E2. The S. cerevisiae structural genes coding for all five enzymes involved in the conversion of xylulose to glycolytic intermediates were also overexpressed: xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase. In addition, the GRE3 gene encoding aldose reductase was deleted to minimise xylitol production. The seven modified genes are listed in FIG. 39. The genetic modifications at the GRE3, RKI1, RPE1, TAL1, and TKL1 loci were designed to leave behind minimal vector DNA and no antibiotic markers. Each locus' DNA was sequenced to confirm the expected results. Each of the seven genetic modifications were sequentially introduced into strain M0086. FIG. 40 shows the progression of modifications from top to bottom together with the designations for the strain at each step in the process, starting with M0086 and finishing with M0407.

[0332] The deletion of GRE3 and the increased expression of RKI1, RPE1, TAL1, and TKL1 involve modifications of the endogenous S. cerevisiae loci. In the case of GRE3, both alleles were deleted. For the other four loci, only a single allele was modified. All of the modifications of endogenous loci required the use of selectable antibiotic markers including kan.sup.r from the Escherichia coli transposon Tn903 (confers resistance to G418). nail from Streptomyces noursei (confers resistance to clonNAT/nourseothricin), and dsdA from Escherichia coli (confers resistance to D-serine.) After selection for a desired genomic modification, the antibiotic marker was excised from the genome using the loxP/cre recombinase system. The cre recombinase was carried on plasmid pMU210 which contains a zeocin resistance marker. Loss of pMU210 as well as all antibiotic markers was tested on the appropriate selective media. Subsequent PCR genotyping and DNA sequencing confirmed removal of the antibiotic markers from the modified genomic loci.

[0333] The overexpression of RKI1, RPE1, TAL1, and TKL1 was achieved by placing the S. cerevisiae triose phosphate isomerase promoter, TP1, immediately 5' of each of the four ORFs. For TAL1 and RKI1, small portions of their endogenous promoters were deleted. To avoid disruption of adjacent ORFs and possible transcriptional regulatory elements, the introduction of the TP1 promoter at the RPE1 and TKL1 loci was done such that the RPE1 and TKL1 loci were duplicated with the duplicate copies of both loci being regulated by the TP1 promoter.

[0334] In order to boost M0407's xylose-utilization and increase its pretreatment inhibitor tolerance, the strain was maintained in a chemostat for four weeks under the following sequential conditions described in Table 10.

TABLE-US-00015 TABLE 10 Conditions to Improve M0407. Duration Residence (days) Time (h) Media 5 24 YPX, 20 g/L xylose 5 18 YPX, 20 g/L xylose 7 24 YPX + 25% of a 30% MS129 washate (21.5 g/L xylose) 14 24 YPX + 75% of a 30% MS129 washate (~22% solids equivalent)

[0335] An aliquot of the adapted chemostat culture was plated on YPXi50% and nine M0407 "adapted" colonies were screened in YPDXi media (100 g/L glucose, 50 g/L xylose, 25% MS149 pressate). M0407 and M0228 (a xylose-utilizing strain created at Mascoma containing XlyA and XKS1 on plasmids) were included as controls. At 24 hours, the glucose had been entirely consumed by all strains. M0407 and M0228 had utilized 30 and 25 g/L of xylose respectively. All nine M0407 "adapted" colonies had utilized more than 44 g/L of xylose. The highest amount of xylose consumed was 48 g/L. This strain was designated M0509.

[0336] 18S rDNA sequencing was used to confirm strain M0509 as Saccharomyces cerevisiae (Kurtzman C P and Robnett, C J; FEMS Yeast Research 3 (2003) 417-432). A 1774 bp fragment spanning the 18S rDNA was amplified from M0509 genomic DNA and sent for sequencing. The 1753 bp of M0509 18S rDNA sequence exhibited a 100% match to the NCBI sequence for S. cerevisiae 18S (nucleotide accession #Z75578).

[0337] Since strain M0509 was obtained by cultivating M0407 in a chemostat for four weeks, the length of cultivation separating the two strains provides a means to asses the stability of the engineered genetic modifications. Comparison of the DNA sequence of M0407 and M0509 at the GRE3, RKI1, RPE1, TAL1, and TKL1 loci showed no changes. This suggests that the genetic modifications at these loci are genetically stable, at least under the growth conditions used.

[0338] Real Time PCR analysis was used to estimate the copy number of integrations of the XylA/XKS1 vector. M0407 has approximately 10 copies of the vector, whereas M0509 has approximately 20 copies. This suggests that the copy number of the XylA/XKS1 vector can be increased by extended cultivation on xylose media.

[0339] To further asses the stability of the XylA/XKS1 integrations, M0509 was cultivated for .about.50 generations in liquid media with either glucose or xylose as the sole carbon source. After 50 generations, an individual colony was isolated from each culture and the number of XylA/XKS1 integrations quantified and compared to the original M0509 freezer stock. The colony isolated from the xylose-culture had .about.20 copies of XylA/XKS1, the same as the freezer stock. The glucose-cultured colony exhibited a slightly decreased copy number, .about.16.

[0340] The slight decrease in XylAlXKS1 copy number of the glucose-colony raises the question of the strain's performance. To partially address this question, xylose consumption was compared between the xylose-isolate, glucose-isolate, and freezer stock. The freezer and xylose-propagated isolates utilized all of the xylose in 24 hours and produced identical amounts of ethanol, but the glucose-propagated strain consumed only half as much xylose. FIG. 20.

Example 13: Selection of a Thermotolerant, Robust, Xylose-Utilizing Strain

[0341] M1105 is capable of fermentation at temperatures above 40.degree. C. in the presence of 8 g/L acetate. M1105 was constructed in a M0509 background and is therefore an industrially robust strain capable of converting both glucose and xylose into ethanol.

[0342] M1105 was isolated following four rounds of selection/adaptation in a cytostat as outlined in FIG. 41 and described as follows. The temperature was increased from 38.degree. C. to 41.degree. C. during the course of the experiment. M1017 (ATCC deposit designation ______, deposited on Nov. 23, 2009)) was isolated from this first cytostat run and was later confirmed by PCR of the GRE3 locus to be a descendant of M0509. M1017 was used to inoculate a second cytostat run using YMX media (yeast nitrogen base, 2 g/L xylose) at 41.degree. C. M1046 was isolated from this second cytostat run. At 42.degree. C. on YPX50, M1046 grew slowly yet with a doubling time 36% shorter than M1017. M1080 was isolated from a cytostat inoculated with M1046 and YMX media at 40.degree. C. M1080 grew with a specific growth rate of 0.22 h.sup.-1 on YMX at 40.degree. C. M1105 was isolated from M1080 based on selection in the cytostat using YPD2X10+acetate media (2 g/L glucose, 10 g/L xylose, 8 g/L acetate, pH 5.4) at 39.degree. C.

[0343] M1105 grows 10-20% faster than M0509 in rich media at 35.degree. C. In addition, M1105 has increased acetate tolerance as the strain can grow more quickly than its ancestral strains in the presence of acetate. FIG. 21. While the parental strains required glucose for tolerance to acetate at high temperatures, M1105 does not require glucose or complex medium components to grow in the presence of 7 g/L acetate at pH 5.4.

[0344] To test fermentation performance, M1105 was inoculated at approximately 0.7 g/L DCW in 18% MS419 using 3.8 mg Zoomerase/g feedstock at 40.degree. C. M1105 produced 3.55% (w/v) ethanol by 168 hours. The time course is presented in FIG. 22 along with a similar run performed with M1088 (described below) for comparison. A similar run using only 0.15 g/L DCW for inoculum resulted in 2.9% (w/v) ethanol and some sugar accumulation during the experiment. FIG. 23.

Example 14: Adaptation of a Thermotolerant, Robust, Xylose-Utilizing Strain

[0345] M1254 is capable of fermentation at temperatures above 40.degree. C. in the presence of 12 g/L acetate, exhibiting an increased robustness relative to the thermotolerant strain M1105.

[0346] M1254 was isolated following three rounds of selection/adaptation in a cytostat as outlined in Table 11 and FIG. 42 and described as follows. The first cytostat run was inoculated with M1105. YMX media (yeast nitrogen base w/o amino acids, 20 g/L xylose) plus 8 g/L acetate was used at pH 5.5 and 40.degree. C. M1155 was isolated from this first cytostat run and used to inoculate a second cytostat containing YPD media (yeast extract, peptone, 20 g/L glucose) plus 12 g/L acetate at pit 5.4 and 41.degree. C. M1202 was isolated from this second cytostat run. M1254 was isolated from a third cytostat run inoculated with both M1155 and M1202 in yeast nitrogen base w/o amino acids+5% solids equivalent MS419 hydrolysate media at pH 4.8, 39.degree. C.

TABLE-US-00016 TABLE 11 Evolutionary Conditions to Generate M1254 from M1105. Parental New Strain(s) Evolutionary Condition Strain M1105 Xylose minimal + 8 g/L acetate, pH 5.5, 40.degree. C. M1155 M1155 Complex glucose + 12 g/L acetate, pH 5.4, 41.degree. C. M1202 M1155 + 5% solids equivalent MS419 hydrolysate + M1254 M1202 yeast nitrogen base w/o amino acids, pH 4.8, 39.degree. C.

[0347] M1254 grows 7.3.+-.0.9% faster than M1202 and 17.+-.2.0% faster than M1155 in 5% solids equivalent MS419 hydrolysate, which is the condition under which strain M1254 was selected. However, standard fermentation medium limits fermentation performance. Accordingly, use of this strain should be with lower ammonium concentrations, such as 1.1 g/L diammonium phosphate (DAP) or lower than 3 g/L DAP. FIG. 24 demonstrates the higher fermentation rate using the lower DAP concentration. The fermentations were performed using 18% MS149, 4 mg external cellulase/g TS, 40.degree. C., 0.5 g/L inoculation DCW M1254 and pH 5.4. The pH was controlled using 5 M potassium hydroxide, and 1 g/L magnesium carbonate was fed with each solids feeding. All enzyme was front loaded, while the solids were fed at five time points (0, 3, 6, 24, and 48 hours) in equal size feedings of 3.6% TS.

[0348] M01360 was created from M1254 using the evolutionary conditions described in Table 12 below.

TABLE-US-00017 TABLE 12 Conditions to Generate M1360 from M1254. Parental New Strain(s) Evolutionary Condition Strain M1254 Complex with low xylose + 8 g/L acetate, pH 5.4, M1339 40.degree. C. M1339 Complex xylose + synthetic inhibitor mixture M1360 (including 8 g/L acetate), pH 5.4, 40.degree. C.

[0349] M1360, while still substantially inhibited by the synthetic inhibitor mixture, grows at 40.degree. C. with a doubling time of approximately 5 hours. FIG. 25. In industrially relevant medium, M1360 is able to generate over 60 g/l ethanol from glucose along with 5 g/L dry cell weight in 48 h at 40.degree. C. beginning with only 60) mg/L dry cell weight. FIG. 26.

[0350] Enzyme activity is known to increase as temperature increases, and thus it is desirable to have thermotolerant S. cerevisiae strains. FIG. 27 shows three equivalent SSFs with 18% PHW solids loaded. The reactions carried out at 40.degree. C. show approximately 17% more ethanol produced than the control reaction carried out at 35.degree. C., when both reactions were carried out at the same external enzyme loading (4 mg/g). This increased performance represents a substantial cost savings for the process.

Example 15: Expression of Cellulases in a Robust Xylose-Utilizing Strain

[0351] M1088 is capable of secreting three distinct cellulolytic enzymes: .beta.-glucosidase from S. fibuligera (SfBGL), cellobiohydrolase 2b from C. lucknowense (C1CBH2b), and cellobiohydrolase I from T. emersonii fused to the T. reesei cellobiohydrolase I cellulose binding domain (TeCBH1+CBDTrCBH1). The M1088 genome also contains genes that encode for polypeptides capable of providing resistance to the following antibiotics: kanamycin, nourseothricin, and hygromycin B. Plasmid pMU624, which is also present in M1088, contains a gene encoding for a polypeptide capable of providing resistance to ampicillin. The steps used to generate M1088 and M0963 from M0509 are summarized in Table 13 below.

TABLE-US-00018 TABLE 13 Strains Used to Generate Strains M1088 and M0963 Strain Genotype Parent Description M0509 gre3::loxP/gre3::loxP TALI+/loxP- PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 RPE1+/loxP-PTPI-RPE1 TKL+/loxP-PTPI-TKL delta::PTPI- xylA PADH1-XKS::delta M0539 URA-3/ura-3::kanMX M0509 A single copy of the genomic URA-3 gre3::loxP/gre3::loxP TALI+/loxP- gene was deleted and replaced with a PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 kanMX cassette. The KanMX gene RPEI+/loxP-PTPI-RPEI cassette provides resistance to TKL+/loxP-PTPI-TKL delta::PTPI- kanamycin (an aminoglycoside xylA PADH1-XKS::delta antibiotic). M0544 ura-3::kanMX/ura-3::kanMX M0539 The second copy of the genomic gre3::loxP/gre3::loxP TAL1+/loxP- URA-3 gene was deleted and PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 replaced with a kanMX cassette. RPE1+/loxP-PTPI-RPE1 TKL+/loxP-PTPI-TKL delta::PTPI- xylA PADH1-XKS::delta M0749 ura-3::kanMX/ura-3::kanMX M0544 A single copy of the genomic FUR-1 gre3::loxP/gre3::loxP TAL1+/loxP- gene was deleted and replaced with a PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 Streptomyces noursei nat1 cassette. RPE1+/loxP-PTPI-RPE1 The nat1 gene cassette provides TKL+/loxP-PTPI-TKL delta::PTPI- resistance to the antibiotic xylA PADH1-XKS::delta nourseothricin/clonNAT (an (pMU782)fur1::nat aminoglycoside antibiotic). M0867 FUR-1/fur-1::nat ura-3::kanMX/ura- M0749 The plasmid pMU624 was 3::kanMX gre3::loxP/gre3::loxP transformed into the strain. pMU624 TAL1+/loxP-PTPI-TAL1 can replicate in S. cerevisiae (2 RKI1+/loxP-PTPI-RKI1 micron ori and URA-3) and E. coli RPE1+/loxP-PTPI-RPE1 (pBMR ori and ampicillin resistance TKL+/loxP-PTPI-TKL delta::PTPI- gene: beta-lactam antibiotic). xylA PADH1-XKS::delta pMU624 also carries the T. emersonii (pMU782)fur1::nat; [pMU624] CBH1 + CBDTrCBH1 gene regulated by the ENO1 promoter and terminator. M0759 fur-1::hyg/fur-1::nat ura- M0867 The second copy of the genomic 3::kanMX/ura-3::kanMX FUR-1 gene was deleted and gre3::loxP/gre3::loxP TAL1+/loxP- replaced with a hygMX cassette. The PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 hygMX gene cassette encodes for a RPE1+/loxP-PTPI-RPE1 hygromycin B phosphotransferase TKL+/loxP-PTPI-TKL delta::PTPI- that confers resistance to hygromycin xylA PADH1-XKS::delta B (an aminoglycoside antibiotic). (pMU782) fur1::nat; [pMU624]; (pMU1037) fur1::hyg M1088 fur-1::hyg/fur-1::nat ura- M0759 Two distinct integration cassettes 3::kanMX/ura-3::kanMX were transformed into the strain and gre3::loxP/gre3::loxP TAL1+/loxP- multiple copies were integrated into PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 the genome at delta site. One RPE1+/loxP-PTPI-RPE1 cassette contained the cellulolytic TKL+/loxP-PTPI-TKL delta::PTPI- genes S. fibuligeria BGL and xylA PADH1-XKS::delta [pMU624] C. lucknowense CBH2b. The other (pMU1260) delta::PGKprom- cassette contained the cellulolytic SfBGL-PGKterm, ENO1prom- genes S. fibuligeria BGL and a TeCBH + TrCBD-ENO1term T. emersonii chimeric CBH1. (pMU1169) delta::PGKprom- SfBGL-PGKterm, ENO1prom- ClCBH2-ENO1term M0963 fur-1::hyg/fur-1::nat ura- M0759 Linear DNA from the 4 plasmids 3::kanMX/ura-3::kanMX shown was transformed into M0759. gre3::loxP/gre3::loxP TAL1+/loxP- 24 of the resulting colonies were then PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 passaged for a week in YPD media RPE1+/loxP-PTPI-RPE1 containing zeocin at a low level (50 TKL+/loxP-PTPI-TKL delta::PTPI- ug/mL), and assayed. The resulting xylA PADH1-XKS::delta strain M0963 was the best of those (pMU782) fur1::nat; [pMU624]; found in the avicel assay. (pMU1037) fur1::hyg; (pMU755) delta::ZeoMX, ENO1prom-TeCBH1w/TrCBD- ENO1term; (pMU809) delta::ZeoMX, ENO1prom- ClCBH2b-ENO1term; (pMU663) delta::ZeoMX, ENO1prom-CfEG-ENO1term; (pMU864) delta::ZeoMX, ENO1prom-SfBGL-ENO1term

Example 16: Selection of an Endogluconase for Expression in a Robust Xylose-Utilizing Strain

[0352] Endoglucanases augment the activity of cellobiohydrolases, and therefore, the ability of family 5 endoglucanases to complement the previously identified CBH1 and CBH2 was investigated. Five family 5 endoglucanses were selected and cloned under control of the ENO1 promoter/terminator using the pRDH122 expression plasmid as shown in Table 14.

TABLE-US-00019 TABLE 14 Family 5 endoglucanases expressed in S. cerevisiae. Theoretical Expression enzyme Organism & Gene: CBM domain: plasmid: size Da* Aspergillus kawachii C-terminal CBM1 pRDH145 55034.58 egA Heterodera schachtii C-terminal CBM2 pRDH146 43739.46 eng1 Hypocrea jecorina N-terminal CBM1 pRDH147 44226.91 (anamorph: Trichoderma reesei) eg2 Orpinomyces sp. PC-2 2x C-terminal pRDH148 53103.40 celB CBM10 Irpex lacteus en1 N-terminal CBM1 pRDH149 42357.15

[0353] All plasmids expressing the 5 new EG2-type cellulases were transformed to Y294 (a lab strain) and M0749 (robust xylose utilizing strain; described above) and transformants were confirmed via PCR. FIG. 28 shows several of the M0749 strains that were spotted on SC.sup.-URA plates containing 0.2% of either CMC or lichenin or barley-.beta.-glucan. As can be seen in FIG. 28, the M0749 reference strain yielded small zones on the CMC containing plates. Both pMU471 (Coptotermes formosanus EG) and pRDH147 based strains yielded very good clearing zones on all the tested substrates.

[0354] Along with the reference strain and a strain expressing the Coptotermes formosanus EG (pMU471), the live eg2 expressing strains were tested for avicel and PASC hydrolysis while the cbh2 expressing strains were tested for activity on avicel. The strains were grown in double strength SC.sup.-URA medium (3.4 g/L YNB; 3 g/L amino acid dropout pool without uracil; 10 g/L ammonium sulfate; 20 g/L glucose) that was buffered to pH 6 (20 g/L succinic acid; 12 g/L NaOH, set pH to 6 with NaOH). 10 mL Cultures in 125 mL Erlenmeyer flasks were grown at 30.degree. C. for three days. Three flasks were inoculated for each strain. After incubation, samples were taken for gel analysis and activity measurement. After centrifugation of the samples, 12 .mu.l of each was taken, added to 5 .mu.l of protein loading buffer and boiled for 5 minutes. The samples were subsequently loaded on a 10% SDS-PAGE and separated, followed by silver staining. The results are shown in FIG. 29. Not all strains produced visible bands in the expected size range. The C.f.EG appeared as a band of about 55 kDa as previously seen but the band produced by M0749 seems to be slightly larger than the one produced by Y294. No bands were visible for the H. schachtii eng1, Orpinomyces celB, or L. lacteus en1 products. The H. jecorina EG2 produced by Y294 and M0749 was visible as .about.57 kDa bands. The increased weight compared to the predicted 44 kDA size may represent hyperglycosylation. The A. kawachii EGA produced by Y294 was visible as a .about.42 kDa band. However, the A. kawachii EGA produced by M0749 was clearly visible as a .about.120 kDa band. The extra weight may signify hyperglycosylation.

[0355] All strains were tested for activity using the high-throughput avicel conversion method as prescribed. Strains expressing endoglucanases were also tested for activity on PASC. The DNS used for the assay procedure contained phenol which, according to literature, renders greater sensitivity. Activity data can be seen in FIG. 30.

[0356] The M0749 strain expressing H.j.eg2 (pRDH147) produced the highest levels of secreted activity as measured on PASC or avicel of the EG2s tested. The activity of this enzyme was higher on PASC and avicel than C.f.EG (pMU471). The synthetic A.k.EGA (pRDH145) also gave appreciable activity on both substrates. This product seems to have been produced at higher levels in M0749 than in Y294 and yielded greater activity than C.f.EG on avicel and PASC when produced in this strain.

Example 17: Expression of an Endogluconase in Robust Xylose-Utilizing Yeast

[0357] Several strains were created to test the impact of co-expressing TrEG2 with CBHs in a robust xylose utilizing strain background. M1088 was transformed with a construct to integrate TrEG2 at the rDNA locus using the Sh-ble gene as a marker (pMU1409). A similar transformation was done, but integrating TeCBH1w/TrCBD to increase the copy number of that gene. 43 transformants from both transformations along with duplicate M1088 cultures were grown in 20 ug/mL zeocin containing YPD and the avicel assay was performed. FIG. 31 shows the results of those assays. The data show that a very large proportion of strains transformed with the TrEG2 construct had significantly increased avicel conversion ability, while transformants with additional TeCBH1w/TrCBD copies had only marginal improvements in avicel hydrolysis.

[0358] Of the strains assayed, the top 9 candidates were chosen and restreaked for single colonies. These single colonies were then grown in YPD with 2 transfers to equal a total of 18 generations. The final transfer (passaged data in FIG. 32) was compared to the first YPD culture (original data in FIG. 32). The data confirms that there is an .about.50% increase in ability of the yeast supernatant to convert avicel when TrEG2 is overexpressed.

[0359] In addition, strain M1403, which contains heterologous genes encoding S. fibuligera (SfBGL), cellobiohydrolase 2b from C. lucknowense (C1CBH2b), cellobiohydrolase I from T. emersonii fused to the T. reesei cellobiohydrolase I cellulose binding domain (TeCBH1+CBDTrCBH1), and Heterodera schachtii eng1 was produced in the M1254 background. Strain M1284, which contains heterologous genes encoding those same four cellulases was produced in the M0509 background. Strains M1284 and M1403 are described in more detail in Table 15.

TABLE-US-00020 TABLE 15 Endogluconase Expressing Yeast Strains. Strain Genotype Parent Description M1403 (pMU1339) delta::MET3prom- M1254 Linear DNA cassettes created by SfBGL-PGKterm, ENO1prom- restriction digests of plasmids were TeCBH1 + TrCBD-ENO1term integrated in multiple copies into the (pMU1260) delta::PGKprom-SfBGL- genome at the Ty1 delta sites and PGKterm, ENO1prom- rDNA sites. TeCBH + TrCBD-ENO1term (pMU1169) delta::PGKprom-SfBGL- PGKterm, ENO1prom-ClCBH2- ENO1term (pMU1409) rDNA::ZeoMX, ENO1prom-HjEG2- ENO1term M0991 gre3::loxP/gre3::loxP TAL1+/loxP- M0509 A single copy of the genomic LEU-2 PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 gene was deleted and replaced with a RPE1+/loxP-PTPI-RPE1 hygMX cassette. TKL+/loxP-PTPI-TKL delta::PTPI- xylA PTPI-XKS LEU2/leu2D::hph M0992 gre3::loxP/gre3::loxP TAL1+/loxP- M0991 The second copy of the genomic PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 LEU-2 gene was deleted and RPE1+/loxP-PTPI-RPE1 replaced with a Streptomyces noursei TKL+/loxP-PTPI-TKL delta::PTPI- nat1 cassette. xylA PTPI-XKS leu2D::hph/ leu2D::nat M1162 gre3::loxP/gre3::loxP TAL1+/loxP- M0992 A linear DNA cassette created by PTPI-TAL1 RKI1+/loxP-PTPI-RKI1 restriction digests of plasmid RPE1+/loxP-PTPI-RPE1 pMU1379 was integrated in multiple TKL+/loxP-PTPI-TKL delta::PTPI- copies into the genome at the Ty1 xylA PTPI-XKS leu2D::hph/ delta sites. leu2D::nat (pMU1379) delta::leu2-19, ENO1prom-TeCBH + TrCBD- ENO1term M1284 gre3::loxP/gre3::loxP TAL1+/loxP- M1162 Linear DNA cassettes created by PTP1-TAL1 RKI1+/loxP-PTPI-RKI1 restriction digests of plasmids RPE1+/loxP-PTPI-RPE1 pMU1169 and pMU1409 were TKL+/loxP-PTPI-TKL delta::PTPI- integrated in multiple copies into the xylA PTPI-XKS leu2D::hph/ genome at the Ty1 delta sites and leu2D::nat rDNA sites. (pMU1379) delta::leu2-19, ENO1prom-TeCBH + TrCBD- ENO1term (pMU1169) delta::PGKprom-SfBGL-PGKterm, ENO1prom-ClCBH2-ENO1term (pMU1409) rDNA::ZeoMX, ENO1prom-HjEG2-ENO1term

Example 18: Conversion of Lignocellulosic Substrates Via CBP Yeast Strains

[0360] Expression of cellulases in yeast, particularly CBH1 (T. emersonii CBH1 w/ T. reesei CBD attached), CBH2 (C. lucknowense CBH2b), EG2 (T. reesei EG2), and BGL (S. fibuligera BGL) dramatically reduces the need for externally added enzymes during enzymatic conversion of lignocellulose to ethanol. To test the effect of overexpressing these enzymes, several strains were constructed and tested on a number of substrates.

[0361] FIG. 33 presents data from a CBP fermentation of paper sludge by an engineered thermotolerant S. cerevisiae host strain (parent strain M1254, cellulolytic derivative M1403). The data for M1254 alone demonstrates that the addition of cellulase (i.e. zoomerase) is required for ethanol production from paper sludge. The data for M1430 where no external cellulase is added (filled orange squares), demonstrates that this strain can convert a substantial fraction (.about.80%) of the "convertible" substrate by virtue of its expressed cellulases. Fermentations with additional external cellulase added to the M1403 strain demonstrate the ultimate potential of enzymatic conversion for the paper sludge substrate. Visual inspections demonstrated that the non-CIP strain was not able to liquefy the substrate, whereas the CBP strain was.

[0362] Furthermore, the CBP strain M1179, which expresses CBH1, CBH2, EG2, and BGL can convert paper sludge to a large extent without added cellulase enzyme. FIG. 34. The control strain in this reaction, M0509, made only a small amount or ethanol during this reaction. The data also show that M1179 can convert this material when loaded at lower cell density (1 g/L) as opposed to the higher cell density (10 g/L) used in other reactions. This implies that the strain is able to grow and produce cellulase throughout the fermentation experiments.

[0363] Pretreated hardwood (PHW) can also be converted by CBP strains. FIG. 35, shows the effect of using a cellulase expressing strain (M0963), compared to a control strain not expressing cellulases (M0509) during fermentation of PHW. The comparison demonstrates that the CBP strain can achieve the same yield of ethanol from PHW when only 2 mg/g of external enzyme are loaded compared to when 4 mg/g of M0509 are loaded in the process. This 2-fold reduction in external enzyme needed represents a large potential cost reduction in the process.

[0364] CBP strains are capable of producing high ethanol titers from PHW as well. FIG. 36 shows that a 30% washed solids fermentation can generate titers of ethanol up to about 70 g/L with minimal external enzyme loaded 4 mg/g and a relatively low cell inoculum (2 g/L). The ability of the low cell density cultivation to eventually catch up to and pass the high cell density culture indicates that the strain grows and continues to make enzyme throughout the fermentation.

[0365] In addition to PHW, corn stover has been implicated as good substrate for conversion to ethanol via an enzymatic saccharification. FIG. 37 demonstrates that pretreated corn stover can be converted well by CBP yeast strains. The CBP strain in this experiment was able to convert about 82% of what was converted with a high enzyme loading (15 FPU, or about 20 mg/g) could achieve. The non-CBP strain made about 60% of the ethanol that the CBP strain was able to achieve.

Example 19: Comparison of CBH1 Cellulases

[0366] In order to provide additional data on the expression levels of different CBH1 enzymes, selected strains were grown in YPD-medium and activities on MULac and Avicel were assayed. Both Y294 and M0749 transformants were studied, and the results are shown in FIG. 38.

[0367] These examples illustrate possible embodiments of the present invention. While the invention has been particularly shown and described with reference to some embodiments thereof, it will be understood by those skilled in the art that they have been presented by way of example only, and not limitation, and various changes in form and details can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0368] All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

Sequence CWU 1

1

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cgaaaaccac ccatccttga cttggcaaca atgttcctct 120ggtggttctt gtactactca aaacgggaag gttgttattg acgctaactg gagatgggtt 180cacactacct ccggttacac caactgttac actggtaaca cttgggatac ttccatctgt 240ccagacgacg ttacctgtgc tcaaaactgt gctttggacg gtgctgacta ctccggtact 300tacggtgtca ctacctctgg caacgcgttg agattgaact tcgtcaccca atcttctggt 360aagaacatcg gttctagatt gtacttgttg caagacgata ctacttacca aatcttcaag 420ttgttgggtc aagagttcac tttcgacgtt gatgtttcca acttgccttg tggtttgaac 480ggtgctttgt acttcgttgc tatggacgcc gacggtaact tatccaagta cccaggtaac 540aaggccggtg ccaagtacgg taccggttac tgtgattctc aatgtccaag agacctaaaa 600ttcattaacg gtcaagctaa cgtcgaaggt tggcaaccat ctgctaacga tccaaacgcc 660ggtgtcggta atcacggttc ctcctgtgct gaaatggacg tttgggaagc taactctatc 720tccaccgccg tcactccaca tccatgtgat accccaggtc aaaccatgtg tcaaggtgat 780gattgtggtg gtacctactc ttccactaga tacgctggta cctgtgacac cgacggttgt 840gatttcaacc cataccaacc aggtaaccac tctttctacg gtccaggtaa gattgtcgat 900acttcttcta agttcactgt tgtcactcaa ttcattaccg acgatggtac cccatctggt 960accctaactg aaattaagag attctacgtc caaaacggta aagtcattcc acaatccgaa 1020agcaccattt ccggtgttac cggtaactcc atcaccactg aatactgtac cgctcaaaag 1080gccgcctttg acaacaccgg tttcttcacc catggtggtt tgcaaaagat ttctcaagcc 1140ttggctcaag gtatggtttt ggtcatgtcc ttgtgggatg accacgctgc taacatgttg 1200tggttggatt ctacttaccc aactgacgct gatccagaca ccccaggtgt tgctagaggt 1260acttgtccaa ccacttctgg tgttccagct gacgtcgaat ctcaaaaccc taactcttac 1320gttatctact ctaacatcaa ggtgggtcca attaactcca ccttcactgc taactaactc 1380gag 138331380DNATalaromyces emersonii 3gaattcatgc taagaagagc tttactattg agctcttctg ctatcttggc cgttaaggct 60caacaagccg gtaccgctac tgctgaaaac caccctccat tgacctggca agaatgtacc 120gctccaggtt cttgtaccac ccaaaacggt gctgtcgtct tggacgctaa ctggagatgg 180gtccacgacg tcaacggtta cactaactgt tacaccggta acacctggga cccaacttac 240tgtccagacg acgaaacttg cgctcaaaac tgtgccttgg acggtgctga ctacgaaggt 300acttacggtg ttacctcctc tggttcttcc ttgaagttga acttcgtcac tggttctaac 360gtcggttcca gattgtattt gttgcaagat gactccactt accaaatctt caagttgttg 420aacagagaat tttctttcga cgtcgatgtg tccaacttgc cttgtggttt gaacggtgct 480ctatacttcg ttgctatgga cgctgatggt ggtgtttcca agtacccaaa caacaaggct 540ggtgccaaat acggtactgg ttactgtgac tctcaatgtc cacgtgactt gaagtttatt 600gatggtgaag ctaatgtcga aggttggcaa ccatcttcta acaacgctaa cactggcatc 660ggtgaccacg gttcttgctg tgccgaaatg gacgtttggg aagccaactc catttccaac 720gccgtcactc cacacccatg tgacactcca ggtcaaacta tgtgttccgg cgatgactgt 780ggtggtactt actctaacga tagatacgct ggtacctgtg atccagacgg ttgcgacttc 840aatccataca gaatgggtaa cacttccttt tacggtccag gcaagatcat cgacactact 900aagccattca ctgttgtcac ccaattcttg accgacgatg gtactgatac cggtactttg 960tccgaaatca agagattcta catccaaaac tctaacgtca tcccacaacc aaattccgac 1020atctctggtg tcactggtaa ctccattacc accgaatttt gtaccgccca aaagcaagct 1080ttcggtgaca ccgacgactt ctctcaacac ggtggtttgg ctaagatggg tgctgctatg 1140caacaaggta tggttttggt catgtctttg tgggacgact acgctgctca aatgttgtgg 1200ttggactccg attacccaac cgatgccgac ccaaccaccc ctggtatcgc tagaggtacc 1260tgtccaactg actctggtgt tccatctgac gtcgaatccc aatctccaaa ctcctacgtc 1320acttactcca acattaaatt cggtccaatc aactccactt tcactgcttc ttaactcgag 138041392DNATalaromyces emersonii 4gaattcatgc gtaacttgtt ggccttggct ccagccgctt tgttggttgg tgctgccgaa 60gctcaacaat ccttgtgggg tcaatgcggt ggttcctcct ggactggtgc aacttcctgt 120gccgctggtg ccacctgttc caccattaac ccatactacg ctcaatgtgt tccagccact 180gccactccaa ctaccttgac taccaccact aagccaacct ccaccggtgg tgctgctcca 240accactccac caccaactac taccggtact accacctctc cagtcgtcac cagacctgcc 300tccgcctccg gtaatccatt cgaaggttat caattgtacg ctaaccctta ctacgcttct 360gaagtcattt ccttggctat cccatctttg agctccgagt tggtcccaaa ggcctccgaa 420gttgctaagg tcccttcatt tgtctggtta gatcaagctg ccaaggttcc atctatgggt 480gattacttga aggatattca atctcaaaac gctgctggtg ctgatccacc aatcgccggt 540attttcgttg tttacgattt gccagataga gactgtgccg ccgctgcttc taacggtgaa 600ttttctatcg ccaacaacgg tgtcgcttta tacaaacaat atatcgattc cattagagaa 660caattaacca cttactccga cgtccatacc atcttggtta tcgaaccaga ctctttggct 720aacgttgtca ctaacttgaa cgttccaaaa tgtgctaacg ctcaagatgc ttacttggaa 780tgtatcaact acgctattac ccaattggac ttgccaaacg ttgctatgta cttggacgct 840ggtcacgccg gttggttggg ttggcaagcc aacttggccc cagctgctca attattcgct 900tctgtttaca agaacgcctc ttccccagcc tctgttagag gtttggctac caacgtggct 960aactacaacg cctggtccat ttctagatgt ccatcctaca ctcaaggtga cgctaactgt 1020gatgaagaag attacgttaa cgctttgggt ccattgttcc aagaacaagg tttcccagct 1080tacttcatca tcgacacttc ccgtaacggt gtcagaccaa ctaagcaatc tcaatggggt 1140gactggtgta acgttattgg taccggtttc ggtgttagac caaccaccga cactggtaac 1200ccattggaag acgctttcgt ttgggtcaag ccaggtggtg aatccgacgg tacctccaac 1260actactagcc cacgttacga ttaccactgt ggtttgtctg acgctttgca accagctcca 1320gaagctggta cctggttcca agcctacttc gaacaattgt tgactaacgc caacccattg 1380ttctaactcg ag 139251449DNAChrysosporium lucknowense 5atggccaaga agttgttcat taccgctgcc ttagctgccg cagtgcttgc tgcaccagtg 60atcgaagaga gacaaaattg cggagccgtc tggacacagt gcggaggcaa cggctggcaa 120ggcccaacat gttgtgcttc tggctcaacg tgcgtggcac agaacgagtg gtattcccag 180tgccttccaa actcccaggt gacttcttca acaaccccca gctcaacgtc tacttcacag 240agatccacaa gtacctcttc tagcacaacc agaagtggct catcctcatc tagcagtacg 300acccctccac ccgtatcaag tcctgtcacg agtatccctg gcggagcaac ctcaacagcc 360agttattccg gcaatccttt ctctggagtg agattatttg caaacgacta ttatagatca 420gaggttcaca accttgcaat tccttctatg acgggaaccc tagccgcaaa ggcttccgcc 480gtagcagaag tccctagttt ccaatggctt gacagaaacg ttacaataga tacacttatg 540gtacagactt tatctcaggt tagagctttg aataaggccg gtgccaaccc accttatgct 600gcccaattag tagtctatga cttgccagat agagactgtg ctgccgcagc ttctaatggt 660gaattttcca tcgcaaatgg cggagctgca aactatagat catacattga tgcaataaga 720aaacacatca ttgagtattc tgatattaga ataatccttg tgattgaacc agactccatg 780gctaatatgg ttaccaacat gaatgtagcc aagtgttcta acgcagcttc cacataccat 840gagctaaccg tatatgcatt aaaacaactg aatctaccta acgttgctat gtacttagat 900gccggtcatg ccggatggtt gggctggcct gcaaatatcc aacccgcagc tgaattgttc 960gctggaatct acaacgacgc cggaaagccc gctgccgtta gaggcttagc cacaaatgtt 1020gcaaattaca acgcttggtc aattgctagt gccccttctt atacctcacc aaatcctaac 1080tacgatgaga aacattacat agaagcattt tccccattgt taaactccgc tggattccct 1140gccagattca tcgtggatac cggtagaaac ggcaaacaac caactggaca acaacaatgg 1200ggagattggt gtaacgtcaa gggaaccggc ttcggcgtca ggcctacggc aaacaccgga 1260cacgagctag tcgacgcttt tgtatgggtt aagccaggtg gcgaaagtga cggaacaagt 1320gacacgagtg ctgcaagata cgattaccac tgtggtctgt ccgacgcttt acagcccgcc 1380cccgaggctg gacaatggtt ccaggcttat tttgaacaat tgttaacgaa cgcaaatcca 1440ccattctaa 144961551DNATalaromyces emersonii 6atgctaagaa gagctttact attgagctct tctgctatct tggccgttaa ggctcaacaa 60gccggtaccg ctactgctga aaaccaccct ccattgacct ggcaagaatg taccgctcca 120ggttcttgta ccacccaaaa cggtgctgtc gtcttggacg ctaactggag atgggtccac 180gacgtcaacg gttacactaa ctgttacacc ggtaacacct gggacccaac ttactgtcca 240gacgacgaaa cttgcgctca aaactgtgcc ttggacggtg ctgactacga aggtacttac 300ggtgttacct cctctggttc ttccttgaag ttgaacttcg tcactggttc taacgtcggt 360tccagattgt atttgttgca agatgactcc acttaccaaa tcttcaagtt gttgaacaga 420gaattttctt tcgacgtcga tgtgtccaac ttgccttgtg gtttgaacgg tgctctatac 480ttcgttgcta tggacgctga tggtggtgtt tccaagtacc caaacaacaa ggctggtgcc 540aaatacggta ctggttactg tgactctcaa tgtccacgtg acttgaagtt tattgatggt 600gaagctaatg tcgaaggttg gcaaccatct tctaacaacg ctaacactgg catcggtgac 660cacggttctt gctgtgccga aatggacgtt tgggaagcca actccatttc caacgccgtc 720actccacacc catgtgacac tccaggtcaa actatgtgtt ccggcgatga ctgtggtggt 780acttactcta acgatagata cgctggtacc tgtgatccag acggttgcga cttcaatcca 840tacagaatgg gtaacacttc cttttacggt ccaggcaaga tcatcgacac tactaagcca 900ttcactgttg tcacccaatt cttgaccgac gatggtactg ataccggtac tttgtccgaa 960atcaagagat tctacatcca aaactctaac gtcatcccac aaccaaattc cgacatctct 1020ggtgtcactg gtaactccat taccaccgaa ttttgtaccg cccaaaagca agctttcggt 1080gacaccgacg acttctctca acacggtggt ttggctaaga tgggtgctgc tatgcaacaa 1140ggtatggttt tggtcatgtc tttgtgggac gactacgctg ctcaaatgtt gtggttggac 1200tccgattacc caaccgatgc cgacccaacc acccctggta tcgctagagg tacctgtcca 1260actgactctg gtgttccatc tgacgtcgaa tcccaatctc caaactccta cgtcacttac 1320tccaacatta aattcggtcc aatcaactcc actttcactg cttctaaccc tccaggtggt 1380aacagaggta ctaccactac tcgtaggcca gctactacaa ctggttcttc cccaggccca 1440acccaatccc actacggtca atgtggtggt atcggttact ctggtccaac cgtctgtgct 1500tctggtacta cctgtcaagt tttaaaccca tactactctc aatgtttgta g 155171608DNATrichoderma reesei 7atggtctcct tcacctccct gctggccggc gttgccgcta tctctggtgt cctagcagcc 60cctgccgcag aagttgaacc tgtcgcagtt gagaaacgtg aggccgaagc agaagctcaa 120tccgcttgta ccctacaatc cgaaactcac ccaccattga cctggcaaaa gtgttctagc 180ggtggaactt gtactcaaca aactggttct gttgttatcg acgctaactg gagatggaca 240cacgccacta actcttctac caactgttac gacggtaaca cttggtcttc cactttatgt 300ccagataacg aaacttgtgc taagaattgc tgtttggacg gtgccgccta cgcttctacc 360tacggtgtta ccacctccgg taactccttg tctattggtt tcgtcactca atccgctcaa 420aagaacgttg gtgctagatt gtacttgatg gcttctgaca ctacttatca agaatttact 480ttgttgggta acgaattttc tttcgatgtt gacgtttccc aattgccatg tggcttgaac 540ggtgctttgt actttgtctc tatggatgct gacggtggtg tttctaagta cccaactaac 600actgccggtg ctaagtacgg tactggttac tgtgattctc aatgtccacg tgacttgaag 660ttcattaacg gtcaagccaa cgtcgaaggt tgggaaccat cctccaacaa cgctaacacc 720ggtatcggtg gtcacggttc ctgttgttcc gaaatggaca tctgggaagc taacagtatt 780tctgaagctt tgacaccaca cccatgcacc actgtcggtc aagaaatttg tgaaggtgat 840ggatgtggtg gaacctactc tgataacaga tacggtggta cttgtgaccc agacggttgt 900gactggaacc catacagatt gggtaacact tctttctatg gtccaggttc ttctttcacc 960ttggatacca ccaagaagtt gactgttgtt acccaattcg aaacttctgg tgctatcaac 1020agatactacg ttcaaaacgg tgtcaccttc caacaaccaa acgctgaatt gggttcttac 1080tctggtaatg aattgaacga cgactactgt accgctgaag aagctgaatt tggtggttcc 1140tctttctccg acaagggtgg tttgacccaa ttcaagaagg ctacctccgg tggtatggtt 1200ttggttatgt ccttgtggga tgattactac gcaaacatgt tatggttaga cagtacttac 1260ccaactaacg aaacctcctc tactccaggt gctgtcagag gttcctgttc tacctcttct 1320ggtgttccag ctcaagttga atctcaatct ccaaacgcta aggtcacttt ctccaacatc 1380aagttcggtc caatcggttc cactggtaat ccatctggtg gaaaccctcc aggtggtaac 1440agaggtacta ccactactcg taggccagct actacaactg gttcttcccc aggcccaacc 1500caatcccact acggtcaatg tggtggtatc ggttactctg gtccaaccgt ctgtgcttct 1560ggtactacct gtcaagtttt aaacccatac tactctcaat gtttgtaa 160881479DNATrichoderma reesei 8atggtctcct tcacctccct gctggccggc gttgccgcta tctctggtgt cctagcagcc 60cctgccgcag aagttgaacc tgtcgcagtt gagaaacgtg aggccgaagc agaagctgtc 120ccattagaag aaagacaagc ctgctcctct gtttggggtc aatgtggtgg tcaaaactgg 180tctggtccaa cttgttgtgc ttccggttct acctgtgttt actccaacga ctactattcc 240caatgtttgc caggtgctgc ttcctcttcc tcttcaacta gagctgcttc tacaacttct 300agggtctccc caaccacttc cagatcctct tctgctactc caccaccagg ttctactacc 360actagagttc caccagtcgg ttccggtact gctacttact ctggtaaccc tttcgtcggt 420gttactccat gggctaacgc ttactacgct tctgaagttt cttctttggc tatcccatct 480ttgactggtg ctatggctac cgctgctgct gctgtcgcca aagttccatc cttcatgtgg 540ttggacacct tggacaaaac tccattaatg gaacaaacct tggcagacat aaggactgct 600aacaagaacg gcggtaacta cgctggtcaa tttgttgtgt acgacttgcc agacagagac 660tgtgctgctt tggcttccaa cggtgaatac tccatcgctg acggtggtgt cgccaagtac 720aagaactaca ttgataccat tagacaaatc gttgtcgaat actctgacat cagaaccttg 780ttagtcatcg aaccagattc tttagccaat ttagtcacca acttgggtac tccaaagtgt 840gctaacgctc aatctgccta cttagaatgt atcaattatg cagttaccca attgaacttg 900ccaaacgttg ctatgtactt ggacgctggt cacgccggtt ggttgggttg gccagctaac 960caagacccag ccgctcaatt attcgccaac gtttacaaga atgcctcttc tcctagagcc 1020ttgcgtggtt tggctactaa cgtcgctaac tacaacggtt ggaacatcac ttctccacca 1080tcttacaccc aaggtaacgc tgtttacaac gaaaagttgt acattcacgc tatcggtcca 1140ttattggcta accatggttg gtctaacgcc ttcttcatca ccgaccaagg tagatccggt 1200aaacaaccaa ctggtcaaca acaatggggt gattggtgta acgtcatcgg tactggtttc 1260ggtatcagac catccgctaa cactggtgat tccttgttgg attccttcgt ctgggttaag 1320ccaggtggtg aatgtgatgg cacctctgat tcctctgctc caagattcga ttcccactgc 1380gccttgccag acgctttgca accagcccca caagctggtg catggttcca agcttacttt 1440gtccaattgt tgaccaacgc taacccatct ttcttgtaa 147991618DNAChaetomium thermophilum 9ttaattaaac aatgatgtac aagaaatttg cagccctagc tgctttagtt gcaggagctt 60ccgctcaaca ggcatgttca ttgactgccg aaaatcatcc atccttaacg tggaagagat 120gcacgtcagg aggttcatgc tccactgtaa acggagctgt cacaatagat gcaaattgga 180gatggaccca cactgtgtcc ggtagtacaa actgctacac cggtaatcaa tgggatacgt 240ctttgtgtac agatggaaag tcatgcgctc agacctgttg cgtggatgga gcagactact 300cttctactta cggaatcacg acatcaggtg acagtcttaa tttgaaattc gtaaccaagc 360accagtacgg aacaaatgta ggctccagag tgtacttaat ggagaacgat accaaatatc 420aaatgttcga gttattaggc aatgagttta cctttgacgt agacgttagc aatttgggtt 480gcggattaaa cggcgccctt tacttcgtgt ctatggatgc tgacggaggt atgtcaaagt 540attctggtaa caaagccgga gcaaagtacg gtacaggtta ttgtgacgct cagtgcccta 600gagatttgaa gtttatcaac ggagaagcca acgttggtaa ctggacgcca agtactaacg 660acgcaaacgc tggattcggc agatacggta gttgttgctc agaaatggac gtgtgggagg 720ccaataacat ggcaaccgct tttactcctc acccatgtac aactgttgga caatctagat 780gtgaagccga cacgtgcggt ggcacctaca gtagcgatag gtatgcagga gtatgtgatc 840ctgacggttg cgatttcaat gcttatagac aaggagacaa aacgttttat ggtaaaggta 900tgaccgtcga tactaacaag aagatgactg tggttaccca gttccacaag aactcagctg 960gagtattgtc tgaaattaaa agattctacg tccaggatgg aaagattatt gctaatgccg 1020agagtaagat accaggtaac cctggaaata gtatcacaca ggaatactgt gacgctcaga 1080aggtagcttt tagcaacacc gatgacttca atagaaaggg tggaatggct caaatgagta 1140aggctttagc cggtccaatg gtgttggtga tgtctgtttg ggatgatcac tatgcaaaca 1200tgctttggct tgacagcacc tatcctatcg accaagccgg agccccaggt gctgaaaggg 1260gtgcatgtcc aaccacgagt ggtgtgcccg ccgagattga agctcaagtg cctaatagta 1320acgttatctt ttccaatata agattcggac caatcggatc cactgttcca ggtttggatg 1380gatctaatcc tggcaaccca acaaccacgg tagtccctcc agcttcaact tccacaagta 1440gaccaacaag ttcaacgtcc agtccagtgt ctactcctac cggacaacca ggaggctgta 1500ccactcagaa atggggtcaa tgcggtggaa ttggctatac aggttgtacg aattgcgttg 1560caggaaccac ttgtacacag ttaaaccctt ggtactcaca atgcctataa ggcgcgcc 161810969DNACoptotermes lacteus 10atgagatttc cttccatatt caccgctgtt ttgttcgcag cctcaagtgc tttagcagaa 60tgtactaagg gtggatgtac taacaagaat ggatacatag ttcatgataa gcacgtcggt 120gacatccaga atagagacac tttggaccct ccagacttag attatgaaaa ggacgtggga 180gtaaccgtgt ccggtggaac ccttagtcaa agattagtct caacttggaa cggtaagaaa 240gtcgtgggaa gtagattgta tattgtggac gaagccgacg agaaatatca attattcaca 300tttgtcggta aggagttcac ctataccgtt gatatgtccc agatccaatg tggaatcaat 360gccgcattat acacagtgga aatgcctgcc gctggaaaga cccctggagg tgttaagtat 420ggatatggat attgtgatgc caactgcgtg gatggagatt gttgtatgga gttcgatatc 480caagaagctt ctaacaaggc aatcgtttac accacccatt cctgtcaaag tcaaacttca 540ggttgcgata cctcaggatg cggttacaac ccttacagag acagtggtga caaggcattc 600tggggaacaa ctataaacgt aaaccagcct gtgacaattg taacacagtt tatcggttct 660ggtagttcct taactgaagt caaaagattg tgcgtgcaag gtggaaagac cttccctcca 720gccaaatcat taaccgacag ttattgtaat gccaacgact atagaagttt gagaactatg 780ggtgcatcca tggctagagg acacgttgtt gtgttttctt tgtgggattc taatggtatg 840agttggatgg atggaggtaa cgccggtcct tgtacctcat ataatattga atctttggaa 900tccagtcagc caaacttaaa ggtcacatgg tcaaacgtga aatacggaga gatcgattct 960ccttattaa 969111356DNACoptotermes formosanus 11atgagattcc cttccatttt cactgctgtt

ttgttcgcag cctcaagtgc tttagcagcc 60tatgactaca agacagtatt gaagaactcc ttgttgttct acgaagctca aagaagtgga 120aaattgcctg cagaccagaa ggtgacctgg agaaaagatt ccgcattaaa cgacaaggga 180cagaagggag aggacttaac tggaggttat tacgacgccg gagactttgt gaagttcggt 240tttccaatgg catacacagt taccgtgttg gcctggggtt tagtcgatta tgaatctgct 300tacagtactg cgggtgcctt ggatgatggt agaaaggcct tgaaatgggg tacagattat 360ttcttgaaag cacataccgc tgccaatgag ttttacggac aggtgggtca gggagatgtg 420gatcatgctt actggggacg tcctgaggac atgactatgt ctagaccagc ttacaagatc 480gatacatcaa aacctggtag tgacttagct gcagaaacag cagccgcttt agcagcaacc 540gcaatagctt acaagtcagc cgattctacc tacagtaaca acttaattac tcatgcaaag 600cagttgttcg attttgcaaa caattataga ggaaagtact ctgatagtat taccgatgcc 660aagaatttct atgcatccgg tgattataag gacgaattag tatgggctgc agcctggttg 720tatagagcta caaatgataa cacttactta accaaagccg aatcattgta taatgaattt 780ggtttaggat cttggaacgg tgcattcaat tgggataaca agatatccgg agttcaggtc 840ttattagcca aattgacatc caaacaagca tacaaagata aagttcaggg ttatgttgat 900tacttagtct cctctcaaaa gaaaactcca aagggattgg tctatattga ccaatgggga 960accttaagac acgcagctaa tagtgccttg atcgctttac aggccgctga tttgggtata 1020aacgctgcta gttatagaca atacgcaaag aagcaaattg attatgcctt aggtgacgga 1080ggtcgttctt acgtggtcgg attcggaact aaccctccag taagacctca tcatagatcc 1140agttcctgtc ctgacgcacc agccgcttgc gactggaata cttacaactc tgccggacca 1200aatgcccacg tcttgaccgg agccttagta ggtggaccag attccaacga tagttacaca 1260gattcacgtt ctgattatat cagtaacgaa gtcgctactg attacaatgc cggtttccaa 1320tctgcagttg ctggtttgtt gaaagccgga gtataa 1356121356DNANasutitermes takasagoensis 12atgagatttc catctatttt cactgccgtc ttatttgcag cctccagtgc attagcagcc 60tatgattata aacaagtttt gagagattcc ttattgttct acgaagctca gagaagcggt 120agattaccag cagaccagaa ggtcacttgg agaaaagatt cagccttgaa tgatcaggga 180gatcaaggtc aagacttaac cggaggttat tttgacgccg gtgattttgt gaaatttggt 240ttcccaatgg catatactgc taccgtcttg gcctggggtt taatcgattt tgaggcagga 300tacagttccg ctggtgcctt ggatgacggt agaaaagcag taaagtgggc aactgattac 360tttataaagg cccacacttc acagaatgag ttttacggac aagtcggtca gggtgacgct 420gatcacgctt tctggggacg tcctgaagat atgaccatgg ctagaccagc ctacaagatt 480gacaccagca gaccaggtag tgacttagcg ggtgaaaccg cagcggcatt ggcagctgcc 540agtatcgtgt ttagaaatgt tgatggtaca tactctaaca acttacttac tcatgccaga 600caattatttg actttgcaaa taactacaga ggaaaatact cagattccat aaccgacgct 660agaaactttt acgccagtgc agattaccgt gacgaattgg tttgggctgc cgcatggttg 720tacagagcta caaatgacaa cacttacttg aataccgcag aatccttgta tgatgaattt 780ggattgcaga actggggtgg agggttaaac tgggattcaa aggtgtctgg tgtccaggtc 840ttgttagcaa aattgaccaa caaacaggct tacaaagata ctgtgcagtc ttacgtgaat 900tacctgatta ataaccagca aaagacccca aaaggattgt tatacattga tatgtggggt 960acattgagac acgccgcaaa tgctgcattc atcatgttgg aagctgccga gttgggttta 1020tccgcatcat cttacagaca gtttgctcaa actcagatcg actacgcttt gggtgacggt 1080ggaagaagtt tcgtctgtgg ttttggttca aaccctccta caagaccaca tcatcgttct 1140tccagttgcc cgcctgcccc agcaacttgt gactggaata cattcaactc acctgaccca 1200aattaccacg tgttatctgg agctttggta ggaggaccag atcaaaacga taattatgtg 1260gatgatagat ccgactacgt ccataacgaa gtggcaaccg actacaacgc cggatttcag 1320agtgctttgg cagccttagt tgctttgggt tattaa 1356131356DNACoptotermes acinaciformis 13atgagattcc ctagtatttt cactgccgtc ttatttgcag ccagttctgc tttagccgca 60tatgattata ccacagtttt gaaaagttcc ttattgttct acgaagctca aagatccggt 120aagttgccag ccgaccagaa ggtcacttgg agaaaagatt cagcattaga cgataaagga 180aataatggag aggacttaac aggaggttat tatgacgctg gtgattttgt gaagtttggt 240tttcctttag catacaccgc tactgtttta gcctggggtt tggtggacta tgaagcgggt 300tactcatccg ctggagccac agatgacggt agaaaggcag tgaaatgggc aaccgactat 360ttgttgaagg cacatactgc cgctaccgag ttatacggac aggtcgggga cggtgacgcc 420gatcacgcat attggggacg tcctgaagat atgactatgg ctagaccagc atacaagatc 480gacgctagca gaccaggatc tgacttagcg ggtgaaaccg ctgccgcttt agccgctgca 540tccatagttt tcaaaggtgt agattcttca tattctgaca acttgttagc tcacgctaaa 600cagttatttg atttcgctga caattataga ggaaaataca gtgattccat aacacaagct 660tcaaactttt acgcctccgg agattacaaa gacgagttag tctgggctgc cacttggttg 720tacagagcaa ccaacgataa tacatatttg accaaagcag aatccttgta caacgagttc 780ggattaggaa actggaacgg agcctttaat tgggacaaca aggtgtccgg tgttcaggtg 840ttgttagcca aattgacctc caagcaggct tataaagaca ccgttcaagg atacgtcgat 900tatttgatta acaatcagca aaagacccca aagggtttgt tatacataga ccaatggggg 960accttgagac acgcagctaa tgctgcctta ataatcttac aggctgctga tttgggtatt 1020tctgccgaca gttatagaca attcgcaaag aagcaaatag attacgcttt aggtgacgga 1080ggtagatcat atgtagttgg ttttggagac aatcctccaa cacatcctca tcaccgttct 1140tcctcatgcc ctgacgcccc agcagtatgc gattggaata ctttcaattc acctgatcca 1200aactttcatg tcttaaccgg agctttagtg ggaggtcctg atcagaacga taactacgtt 1260gatgatcgtt ctgactacgt gtccaacgag gttgcaaccg actataatgc aggattccaa 1320agtgctgtgg ccgctttagt tactttagga gtttaa 1356141356DNAMastotermes darwinensis 14atgagattcc caagtatatt tactgctgtt ttgttcgcag ccagttctgc tttagcagcc 60tatgattaca atgacgtatt aaccaaaagt ttgttgttct acgaagctca aagatccggt 120aagttacctt ctgatcagaa agtcacctgg agaaaagatt cagcattaaa cgataaggga 180caaaatggtg aggacttaac tggtggatat tatgacgccg gtgattacgt gaagtttggt 240tttccaatgg catatactgc taccgttttg gcttggggtt tagtggacca tcctgccgga 300tacagttctg cgggtgtctt ggatgatggt agaaaagctg tgaagtgggt taccgattac 360ttaatcaaag cccacgtatc aaagaacgaa ttatacggac aggtcggtga cggtgacgca 420gatcacgctt attggggacg tccagaggat atgacaatgg caagaccagc atacaaaata 480gacacttcaa gaccaggttc cgacttagcg ggtgaaaccg cagcggcatt ggctgctgca 540tctattgtgt ttaagtcaac agattctaat tacgccaaca ccttattgac ccacgcaaaa 600caattattcg actttgccaa taactataga ggtaagtata gtgattccat aacacaggca 660tctaatttct acagtagttc cgactataaa gatgaattgg tttgggcagc tgtatggttg 720tacagagcca ctaacgatca gacctatttg acaactgcag agaagttata ctcagacttg 780ggattacagt cctggaacgg aggtttcaca tgggacacca aaattagtgg agtagaagtg 840ttattggcta agattactgg taaacaggca tataaggaca aagtaaaggg atattgtgat 900tatatctcag gatctcagca gaaaacacct aaaggattag tttacataga taagtggggt 960tccttaagaa tggccgcaaa cgccgcatat atttgcgctg tagccgcaga cgtcggaatc 1020agttcaacag cttacagaca gttcgccaaa acacagatta attacatatt gggtgatgcc 1080ggacgttctt ttgtggttgg ttacggaaac aacccaccta cacacccaca tcacagatcc 1140agttcatgtc ctgacgcccc agcaacatgc gattggaata actacaacag tgctaaccct 1200aatccacatg ttttatacgg tgcattagtt ggtggaccag attccaacga taattatcaa 1260gacttaagat cagattatgt cgccaacgaa gtggcaacag actacaatgc agccttccag 1320tcattgttag cattaatcgt ggacttaggt ttgtaa 1356151356DNANasutitermes walkeri 15atgagatttc catctatttt cactgccgtc ttatttgcag cctcaagtgc tttagcagcc 60tatgattaca aacaagtatt gagagattcc ttattgttct acgaagctca gagaagcggt 120agattaccag cagaccagaa ggtcacctgg agaaaagatt ccgccttgaa tgatcaggga 180gagcaaggtc aagacttaac cggaggttat tttgacgccg gtgattttgt gaagtttgga 240ttcccaatgg cttatacagc aaccgttttg gcctggggtt taatcgactt tgaagccggt 300tactcttctg ctggtgcctt ggacgatggt agaaaagcag taaagtgggc tactgattac 360tttataaaag cccatacttc tcaaaacgag ttttacggac aagtcggtca gggtgacgta 420gatcacgcat attggggacg tcctgaagat atgacaatgg ctagaccagc ctacaagatt 480gataccagca gaccaggtag tgacttagca ggagaaactg ctgcagcttt ggctgccgca 540tccatcgttt tcaagaatgt agatggtaca tattccaaca acttacttac tcatgctaga 600cagttgtttg atttcgccaa caattacaga ggaaaatact ctgatagtat taccgatgca 660agaaactttt acgctagtgc cgactataga gatgagttag tctgggcagc tgcctggttg 720tacagagcaa ccaacgacaa ttcttacttg aacactgctg aatcattata caacgagttt 780ggattgcaaa attggggtgg agggttaaac tgggattcta aagtgagtgg tgttcaagtt 840ttgttagcca agttgaccaa caaacaagag tataaggaca ctattcaatc atacgtgaat 900tacttaatca ataaccaaca gaaaactcca aagggattgt tatacattga catgtggggg 960accttgagac acgcagctaa cgcagccttt ataatgttag aagctgccga cttaggttta 1020tccgcttcat cttatagaca gttcgcccaa acacaaatag actacgcatt gggggacggt 1080ggacgttctt ttgtctgtgg tttcggttct aatcctccaa ctagacctca tcatagatcc 1140agttcatgcc cgcctgctcc agctacctgt gattggaata cattcaattc tcctgaccca 1200aactacaatg ttttatccgg tgccttggtt ggtggtcctg accagaatga taactacgtg 1260gacgatagaa gtgattatgt ccataatgag gtagcaactg actacaatgc cggtttccaa 1320tcagccttag ccgctttagt cgccttaggt tactaa 1356161356DNAReticulitermes speratus 16atgagattcc caagtatatt tactgccgtc ttatttgcag cctccagtgc attagccgct 60tatgactaca aaacagtatt gtccaattcc ttgttgttct acgaagctca aagatccggt 120aagttacctt ctgaccagaa agtgacctgg agaaaggatt cagcattaaa cgacaaagga 180caaaagggtg aggacttaac cggtggatat tacgacgccg gagactttgt gaaatttggt 240tttccaatgg cttacacagt taccgtattg gcatggggtg ttattgatta cgaatccgcc 300tactctgccg caggagcttt agattcaggt agaaaggcct tgaaatatgg gaccgactat 360ttcttaaagg cacatacagc agctaacgag ttttacggac aggtgggtca aggtgacgtt 420gaccacgcat actggggacg tcctgaagat atgaccatga gcagaccagc atacaaaata 480gacacttcta agcctggttc cgacttagct gcagagactg cagctgcatt agcagccaca 540gctattgcat acaaatctgc cgatgcaaca tattccaaca atttgataac acatgcaaaa 600caattattcg actttgccaa caattacaga ggaaaatatt ccgatagtat taccgatgcc 660aagaactttt atgcttctgg tgattacaaa gacgaattgg tatgggccgc tgcatggttg 720tacagagcaa ccaatgacaa cacatatttg actaaggcag aatccttata caatgaattt 780ggtttgggaa acttcaatgg tgccttcaat tgggataaca aagtctccgg agtccaggtg 840ttattggcca agttaacctc aaaacaagtg tataaggata aggtacagtc ttacgtggac 900tatttgatct cctcacaaaa aaagacacca aaaggtttag tgtacatcga tcaatggggt 960actttaagac acgcagctaa ttctgctttg atcgctttgc aggcagctga cttaggaatt 1020aacgctgcta cttacagagc ctacgcaaag aagcaaatcg actatgcttt gggtgatggt 1080ggaagatcct atgttattgg atttgggacc aaccctccag taagaccaca tcacagaagt 1140tcatcttgcc cagatgcacc agctgtctgc gattggaaca cctataactc cgctggtcca 1200aacgcccacg tgttaaccgg tgcattggtt ggaggacctg atagtaatga tagttatacc 1260gatgctcgtt ctgactacat atccaacgaa gtggcaactg attacaatgc gggtttccaa 1320tccgctgtcg ctggattatt gaaggcgggt gtctaa 1356171227DNANeosartorya fischeri 17atgagatttc catctatttt cactgcagtt ttgttcgcag ccagttccgc tttggcccaa 60cagatcgggt ccatcgccga aaatcatcct gagttgacaa cctatagatg ctcctctcaa 120gctggatgcg tagcacagag tacttccgtc gtgttagata ttaacgctca ttggattcat 180caaaacggtg cccaaacaag ttgcactacc tcaagtggat tggacccttc attgtgccct 240gataaagtca cctgttctca gaactgcgta gtcgaaggaa taaccgacta ctcatctttt 300ggtgtgcaaa actccggaga tgcaatgaca ttaagacagt atcaagttca aaatggacag 360atcaaaacat tgcgtcctag agtgtacttg ttagctgagg atggaatcaa ttactccaaa 420ttgcagttgt tgaaccaaga gtttactttc gatgtggacg cttccaaatt gccttgtggt 480atgaatggag ctttatattt gtcagaaatg gatgcttctg gtggacgttc tgccttgaac 540ccagcgggtg ccacatatgg aacaggttac tgtgatgccc agtgcttcaa cccaggtcca 600tggataaatg gagaagcaaa tactgctgga gccggtgcat gttgccaaga gatggactta 660tgggaagcca actcccgttc taccattttc agtcctcacc catgtacaac tgcgggtttg 720tatgcctgta ctggagctga gtgctactca atctgtgacg gttatggttg cacttacaac 780ccttatgaat taggagccaa agattactat ggttacggtt tgactattga caccgcaaag 840ccaataacag tggttactca gtttatgacc gctgataata cagcaaccgg tacattagca 900gagatcagaa gattatatgt tcaagatggt aaagtaatcg gaaatacagc cgtggccatg 960accgaggcat tttgtagttc tagtagaaca tttgaagagt taggtggttt gcaaagaatg 1020ggagaagctt taggtagagg aatggtgcca gttttctcaa tatgggacga tcctggtttg 1080tggatgcatt ggttagattc tgacggtgca ggaccttgtg gtaatactga aggtgatcct 1140gccttcattc aggctaacta cccaaatacc gccgtaacat tctccaaggt gagatgggga 1200gatatcggtt ctacctatag ttcttaa 122718915DNAReticulitermes flavipes 18atgagatttc catctatttt cactgctgtt ttgttcgcag cctcaagtgc tttagcacaa 60tggatgcaga tcggtggtaa gcagaaatat cctgccttta agccaggtgc taagtacgga 120agaggttatt gtgacggaca gtgccctcac gacatgaagg tgtctagtgg aagagcaaac 180gttgacggat ggaagccaca agacaacgac gaaaatagtg gaaatggaaa attgggtaca 240tgttgctggg agatggatat atgggaagga aacttagtgt cccaagccta caccgttcac 300gctggttcca agtccggaca atatgagtgt actggaacac aatgcggtga caccgacagt 360ggtgaaagat tcaagggaac atgcgataaa gatggttgtg atttcgcaag ttacagatgg 420ggagctacag actattacgg tcctggaaag accgtggaca ccaaacagcc aatgacagtc 480gtgacccagt tcattggtga ccctttgact gagataaaga gagtttatgt acaaggagga 540aaagtcataa acaattccaa aacatctaac ttaggttcag tgtacgattc tttgactgag 600gccttctgcg atgacaccaa acaggttaca ggtgatacaa atgactttaa ggctaaagga 660ggtatgtctg gattctccaa gaacttagac accccacaag ttttggtgat gtctttatgg 720gatgaccata cagctaatat gttatggtta gattctactt atcctaccga tagtacaaag 780ccaggtgccg caagaggtac ttgtgccgtc acctccgggg accctaaaga tgtggaatcc 840aagcaagcca actctcaggt agtttacagt gacattaagt ttggtcctat taattcaaca 900tacaaagcaa attaa 915191428DNATrichoderma reesei 19atggtctcct tcacctccct gctggccggc gttgccgcta tctctggtgt cctagcagcc 60cctgccgcag aagttgaacc tgtcgcagtt gagaaacgtg aggccgaagc agaagctcaa 120caaccaggaa catcaacacc agaagtccat ccaaagttaa caacctataa atgtactaag 180agtggagggt gtgtagcgca ggacacaagt gtggtcttag actggaatta tcgttggatg 240catgatgcca attataattc ctgtactgtt aacggcggtg ttaacactac gttatgcccc 300gatgaagcga cttgtggtaa gaattgtttt attgaagggg ttgactacgc cgctagtggt 360gttacgacga gtgggtcatc cttgacgatg aatcaataca tgccttcttc tagtggtggg 420tattcctctg tgtctccaag gctgtattta ttggattccg atggggaata tgttatgtta 480aaattaaatg ggcaagaact gagttttgat gtggatctat ctgcattacc ttgtggagaa 540aatggtagtc tttatttatc acaaatggac gaaaacggcg gagccaatca gtacaataca 600gctggtgcta attatggttc aggctattgt gatgctcaat gtccagtgca gacttggagg 660aatggcacct taaacacatc acatcaagga ttttgctgta acgaaatgga catattagaa 720ggtaattcaa gagctaatgc actaactccg cactcttgta ctgcgaccgc atgtgattct 780gccggttgtg gtttcaaccc ttatggttct ggttataaga gttactacgg tccgggagac 840accgtggata cgtcaaagac cttcactata atcactcagt ttaacacaga taacggatct 900ccgagtggta atttggtgag tattactagg aaatatcagc agaacggtgt tgatattccg 960tccgcgcagc caggcggtga cactatatct agctgtcctt ccgccagtgc ctatggcgga 1020cttgctacaa tgggtaaggc attgtcctca ggtatggtcc tagtattttc tatttggaat 1080gataattcac aatacatgaa ttggctggat tctggtaatg caggcccttg ctcctctaca 1140gaaggtaacc caagcaatat actagctaat aacccaaata ctcatgttgt ctttagtaat 1200attagatggg gcgatatagg tagcactacg aacagtaccg cacctcctcc tccacctgct 1260agctccacga cattttccac tactagaagg tccagcacta ccagctcatc accatcttgt 1320actcaaaccc attggggaca gtgtggtggt ataggttaca gcggttgcaa aacttgcaca 1380tctggtacta catgccaata cagtaatgac tattactcac aatgttaa 1428202688DNASaccharomycopsis fibuligera 20atggtctcct tcacctccct cctcgccggc gtcgccgcca tctcgggcgt cttggccgct 60cccgccgccg aggtcgaatc cgtggctgtg gagaagcgct cggactcgcg agtcccaatt 120caaaactata cccagtctcc atcccagaga gatgagagct cccaatgggt gagcccgcat 180tattatccaa ctccacaagg tggtaggctc caagacgtct ggcaagaagc atatgctaga 240gcaaaagcca tcgttggcca gatgactatt gttgaaaagg tcaatttgac cactggtacc 300ggttggcaat tagatccatg tgttggtaat accggttctg ttccaagatt cggcatccca 360aacctttgcc tacaagatgg gccattgggt gttcgattcg ctgactttgt tactggctat 420ccatccggtc ttgctactgg tgcaacgttc aataaggatt tgtttcttca aagaggtcaa 480gctctcggtc atgagttcaa cagcaaaggt gtacatattg cgttgggccc tgctgttggc 540ccacttggtg tcaaagccag aggtggcaga aatttcgaag cctttggttc cgacccatat 600ctccaaggta ctgctgctgc tgcaaccatc aaaggtctcc aagagaataa tgttatggct 660tgtgtcaagc actttattgg taacgaacaa gaaaagtaca gacagccaga tgacataaac 720cctgccacca accaaactac taaagaagct attagtgcca acattccaga cagagccatg 780catgagttgt acttgtggcc atttgccgat tcggttcgag caggtgttgg ttctgttatg 840tgctcttata acagagtcaa caacacttac gcttgcgaaa actcttacat gatgaaccac 900ttgcttaaag aagagttggg ttttcaaggc tttgttgttt cggactgggg tgcacaatta 960agtggggttt atagcgctat ctcgggctta gatatgtcta tgcctggtga agtgtatggg 1020ggatggaaca ccggcacgtc tttctggggt caaaacttga cgaaagctat ttacaatgag 1080actgttccga ttgaaagatt agatgatatg gcaaccagga tcttggctgc tttgtatgct 1140accaatagtt tcccaacaga agatcacctt ccaaattttt cttcatggac aacgaaagaa 1200tatggcaata aatattatgc tgacaacact accgagattg tcaaagtcaa ctaccatgtg 1260gacccatcaa atgactttac ggaggacaca gctttgaagg ttgctgagga atctattgtg 1320cttttaaaaa atgaaaacaa cactttgcca atttctcccg aaaaggctaa aagattacta 1380ttgtcgggta ttgctgcagg ccctgatccg ataggttatc agtgtgaaga tcaatcttgc 1440acaaatggcg ctttgtttca aggttggggt tctggcagtg ttggttctcc aaaatatcaa 1500gtcactccat ttgaggaaat ttcttatctt gcaagaaaaa acaagatgca atttgattat 1560attcgggagt cttacgactt agctcaagtt actaaagtag cttccgatgc tcatttgtct 1620atagttgttg tctctgctgc aagcggtgag ggttatataa ccgttgacgg taaccaaggt 1680gacagaagaa atctcacttt gtggaacaac ggtgataaat tgattgaaac agttgctgaa 1740aactgtgcca atactgttgt tgttgttact tctactggtc aaattaattt tgaaggcttt 1800gctgatcacc caaatgttac cgcaattgtc tgggccggcc cattaggtga cagatccggg 1860actgctatcg ccaatattct ttttggtaaa gcgaacccat caggtcatct tccattcact 1920attgctaaga ctgacgatga ttacattcca attgaaacct acagtccatc gagtggtgaa 1980cctgaagaca accacttggt tgaaaatgac ttgcttgttg actatagata ttttgaagag 2040aagaatattg agccaagata cgcatttggt tatggcttgt cttacaatga gtatgaagtt 2100agcaatgcaa aggtctcggc agccaaaaaa gttgatgagg agttgcctga accagctacc 2160tacttatcgg agtttagcta tcaaaatgca aaagacagca aaaatccaag tgatgctttt 2220gctccaacag atttaaacag agttaatgag tacctttatc catatttaga tagcaatgtt 2280accttaaaag acggaaacta tgagtatccc gatggctaca gcactgagca aagaacaaca 2340cctatccaac ctgggggcgg cttgggaggc aacgatgctt tgtgggaggt cgcttataaa 2400gttgaagtgg acgttcaaaa cttgggtaac tccactgata agtttgttcc acagttgtat 2460ttgaaacacc ctgaggatgg caagtttgaa acccctattc aattgagagg gtttgaaaag 2520gttgagttgt ccccgggtga gaagaagaca gttgagtttg agcttttgag aagagatctt 2580agtgtgtggg ataccaccag acagtcttgg atcgttgaat ctggtactta tgaggcctta 2640attggtgttg ctgttaatga tatcaagaca tctgtcctgt ttactatt 268821525PRTHumicola grisea 21Met Arg Thr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Ser Ala 1 5 10 15 Ala Ala Gln Gln

Ala Cys Ser Leu Thr Thr Glu Arg His Pro Ser Leu 20 25 30 Ser Trp Asn Lys Cys Thr Ala Gly Gly Gln Cys Gln Thr Val Gln Ala 35 40 45 Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp Thr His Gln Val Ser Gly 50 55 60 Ser Thr Asn Cys Tyr Thr Gly Asn Lys Trp Asp Thr Ser Ile Cys Thr 65 70 75 80 Asp Ala Lys Ser Cys Ala Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr 85 90 95 Thr Ser Thr Tyr Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 100 105 110 Phe Val Thr Lys Gly Gln His Ser Thr Asn Val Gly Ser Arg Thr Tyr 115 120 125 Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu Leu Leu Gly Asn 130 135 140 Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly Leu Asn 145 150 155 160 Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu Ser Arg 165 170 175 Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185 190 Ala Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile 195 200 205 Glu Gly Trp Thr Gly Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 210 215 220 Tyr Gly Thr Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 225 230 235 240 Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 245 250 255 Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg Tyr Ala 260 265 270 Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly 275 280 285 Asn Lys Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys 290 295 300 Ile Thr Val Val Thr Gln Phe Leu Lys Asp Ala Asn Gly Asp Leu Gly 305 310 315 320 Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Pro Asn Ser 325 330 335 Glu Ser Thr Ile Pro Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp 340 345 350 Cys Asp Arg Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 355 360 365 Lys Gly Gly Met Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val 370 375 380 Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met Leu Trp Leu 385 390 395 400 Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly Ala Glu Arg 405 410 415 Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu 420 425 430 Ala Pro Asn Ser Asn Val Val Phe Ser Asn Ile Arg Phe Gly Pro Ile 435 440 445 Gly Ser Thr Val Ala Gly Leu Pro Gly Ala Gly Asn Gly Gly Asn Asn 450 455 460 Gly Gly Asn Pro Pro Pro Pro Thr Thr Thr Thr Ser Ser Ala Pro Ala 465 470 475 480 Thr Thr Thr Thr Ala Ser Ala Gly Pro Lys Ala Gly Arg Trp Gln Gln 485 490 495 Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 500 505 510 Ile Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 515 520 525 22456PRTThermoascus aurantiacus 22Met Tyr Gln Arg Ala Leu Leu Phe Ser Phe Phe Leu Ala Ala Ala Arg 1 5 10 15 Ala His Glu Ala Gly Thr Val Thr Ala Glu Asn His Pro Ser Leu Thr 20 25 30 Trp Gln Gln Cys Ser Ser Gly Gly Ser Cys Thr Thr Gln Asn Gly Lys 35 40 45 Val Val Ile Asp Ala Asn Trp Arg Trp Val His Thr Thr Ser Gly Tyr 50 55 60 Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr Ser Ile Cys Pro Asp 65 70 75 80 Asp Val Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Ser 85 90 95 Gly Thr Tyr Gly Val Thr Thr Ser Gly Asn Ala Leu Arg Leu Asn Phe 100 105 110 Val Thr Gln Ser Ser Gly Lys Asn Ile Gly Ser Arg Leu Tyr Leu Leu 115 120 125 Gln Asp Asp Thr Thr Tyr Gln Ile Phe Lys Leu Leu Gly Gln Glu Phe 130 135 140 Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala 145 150 155 160 Leu Tyr Phe Val Ala Met Asp Ala Asp Gly Asn Leu Ser Lys Tyr Pro 165 170 175 Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205 Trp Gln Pro Ser Ala Asn Asp Pro Asn Ala Gly Val Gly Asn His Gly 210 215 220 Ser Ser Cys Ala Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Thr 225 230 235 240 Ala Val Thr Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Gln 245 250 255 Gly Asp Asp Cys Gly Gly Thr Tyr Ser Ser Thr Arg Tyr Ala Gly Thr 260 265 270 Cys Asp Thr Asp Gly Cys Asp Phe Asn Pro Tyr Gln Pro Gly Asn His 275 280 285 Ser Phe Tyr Gly Pro Gly Lys Ile Val Asp Thr Ser Ser Lys Phe Thr 290 295 300 Val Val Thr Gln Phe Ile Thr Asp Asp Gly Thr Pro Ser Gly Thr Leu 305 310 315 320 Thr Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Gln 325 330 335 Ser Glu Ser Thr Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu 340 345 350 Tyr Cys Thr Ala Gln Lys Ala Ala Phe Asp Asn Thr Gly Phe Phe Thr 355 360 365 His Gly Gly Leu Gln Lys Ile Ser Gln Ala Leu Ala Gln Gly Met Val 370 375 380 Leu Val Met Ser Leu Trp Asp Asp His Ala Ala Asn Met Leu Trp Leu 385 390 395 400 Asp Ser Thr Tyr Pro Thr Asp Ala Asp Pro Asp Thr Pro Gly Val Ala 405 410 415 Arg Gly Thr Cys Pro Thr Thr Ser Gly Val Pro Ala Asp Val Glu Ser 420 425 430 Gln Asn Pro Asn Ser Tyr Val Ile Tyr Ser Asn Ile Lys Val Gly Pro 435 440 445 Ile Asn Ser Thr Phe Thr Ala Asn 450 455 23455PRTTalaromyces emersonii 23Met Leu Arg Arg Ala Leu Leu Leu Ser Ser Ser Ala Ile Leu Ala Val 1 5 10 15 Lys Ala Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu 20 25 30 Thr Trp Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly 35 40 45 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly 50 55 60 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro 65 70 75 80 Asp Asp Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95 Glu Gly Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn 100 105 110 Phe Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp 115 120 125 Asp Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe 130 135 140 Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 145 150 155 160 Phe Val Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn 165 170 175 Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190 Arg Asp Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln 195 200 205 Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys 210 215 220 Cys Ala Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val 225 230 235 240 Thr Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp 245 250 255 Asp Cys Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp 260 265 270 Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe 275 280 285 Tyr Gly Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val 290 295 300 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu 305 310 315 320 Ile Lys Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn 325 330 335 Ser Asp Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys 340 345 350 Thr Ala Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His 355 360 365 Gly Gly Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu 370 375 380 Val Met Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp 385 390 395 400 Ser Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr Pro Gly Ile Ala Arg 405 410 415 Gly Thr Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln 420 425 430 Ser Pro Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile 435 440 445 Asn Ser Thr Phe Thr Ala Ser 450 455 24459PRTTalaromyces emersonii 24Met Arg Asn Leu Leu Ala Leu Ala Pro Ala Ala Leu Leu Val Gly Ala 1 5 10 15 Ala Glu Ala Gln Gln Ser Leu Trp Gly Gln Cys Gly Gly Ser Ser Trp 20 25 30 Thr Gly Ala Thr Ser Cys Ala Ala Gly Ala Thr Cys Ser Thr Ile Asn 35 40 45 Pro Tyr Tyr Ala Gln Cys Val Pro Ala Thr Ala Thr Pro Thr Thr Leu 50 55 60 Thr Thr Thr Thr Lys Pro Thr Ser Thr Gly Gly Ala Ala Pro Thr Thr 65 70 75 80 Pro Pro Pro Thr Thr Thr Gly Thr Thr Thr Ser Pro Val Val Thr Arg 85 90 95 Pro Ala Ser Ala Ser Gly Asn Pro Phe Glu Gly Tyr Gln Leu Tyr Ala 100 105 110 Asn Pro Tyr Tyr Ala Ser Glu Val Ile Ser Leu Ala Ile Pro Ser Leu 115 120 125 Ser Ser Glu Leu Val Pro Lys Ala Ser Glu Val Ala Lys Val Pro Ser 130 135 140 Phe Val Trp Leu Asp Gln Ala Ala Lys Val Pro Ser Met Gly Asp Tyr 145 150 155 160 Leu Lys Asp Ile Gln Ser Gln Asn Ala Ala Gly Ala Asp Pro Pro Ile 165 170 175 Ala Gly Ile Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala 180 185 190 Ala Ala Ser Asn Gly Glu Phe Ser Ile Ala Asn Asn Gly Val Ala Leu 195 200 205 Tyr Lys Gln Tyr Ile Asp Ser Ile Arg Glu Gln Leu Thr Thr Tyr Ser 210 215 220 Asp Val His Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Val 225 230 235 240 Val Thr Asn Leu Asn Val Pro Lys Cys Ala Asn Ala Gln Asp Ala Tyr 245 250 255 Leu Glu Cys Ile Asn Tyr Ala Ile Thr Gln Leu Asp Leu Pro Asn Val 260 265 270 Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Gln Ala 275 280 285 Asn Leu Ala Pro Ala Ala Gln Leu Phe Ala Ser Val Tyr Lys Asn Ala 290 295 300 Ser Ser Pro Ala Ser Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr 305 310 315 320 Asn Ala Trp Ser Ile Ser Arg Cys Pro Ser Tyr Thr Gln Gly Asp Ala 325 330 335 Asn Cys Asp Glu Glu Asp Tyr Val Asn Ala Leu Gly Pro Leu Phe Gln 340 345 350 Glu Gln Gly Phe Pro Ala Tyr Phe Ile Ile Asp Thr Ser Arg Asn Gly 355 360 365 Val Arg Pro Thr Lys Gln Ser Gln Trp Gly Asp Trp Cys Asn Val Ile 370 375 380 Gly Thr Gly Phe Gly Val Arg Pro Thr Thr Asp Thr Gly Asn Pro Leu 385 390 395 400 Glu Asp Ala Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr 405 410 415 Ser Asn Thr Thr Ser Pro Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp 420 425 430 Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe 435 440 445 Glu Gln Leu Leu Thr Asn Ala Asn Pro Leu Phe 450 455 25482PRTChrysosporium lucknowense 25Met Ala Lys Lys Leu Phe Ile Thr Ala Ala Leu Ala Ala Ala Val Leu 1 5 10 15 Ala Ala Pro Val Ile Glu Glu Arg Gln Asn Cys Gly Ala Val Trp Thr 20 25 30 Gln Cys Gly Gly Asn Gly Trp Gln Gly Pro Thr Cys Cys Ala Ser Gly 35 40 45 Ser Thr Cys Val Ala Gln Asn Glu Trp Tyr Ser Gln Cys Leu Pro Asn 50 55 60 Ser Gln Val Thr Ser Ser Thr Thr Pro Ser Ser Thr Ser Thr Ser Gln 65 70 75 80 Arg Ser Thr Ser Thr Ser Ser Ser Thr Thr Arg Ser Gly Ser Ser Ser 85 90 95 Ser Ser Ser Thr Thr Pro Pro Pro Val Ser Ser Pro Val Thr Ser Ile 100 105 110 Pro Gly Gly Ala Thr Ser Thr Ala Ser Tyr Ser Gly Asn Pro Phe Ser 115 120 125 Gly Val Arg Leu Phe Ala Asn Asp Tyr Tyr Arg Ser Glu Val His Asn 130 135 140 Leu Ala Ile Pro Ser Met Thr Gly Thr Leu Ala Ala Lys Ala Ser Ala 145 150 155 160 Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val Thr Ile 165 170 175 Asp Thr Leu Met Val Gln Thr Leu Ser Gln Val Arg Ala Leu Asn Lys 180 185 190 Ala Gly Ala Asn Pro Pro Tyr Ala Ala Gln Leu Val Val Tyr Asp Leu 195 200 205 Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe Ser Ile 210 215 220 Ala Asn Gly Gly Ala Ala Asn Tyr Arg Ser Tyr Ile Asp Ala Ile Arg 225 230 235 240 Lys His Ile Ile Glu Tyr Ser Asp Ile Arg Ile Ile Leu Val Ile Glu 245 250 255 Pro Asp Ser Met Ala Asn Met Val Thr Asn Met Asn Val Ala Lys Cys 260 265 270 Ser Asn Ala Ala Ser Thr Tyr His Glu Leu Thr Val Tyr Ala Leu Lys 275 280 285 Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala 290 295 300 Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Glu Leu Phe 305 310 315 320 Ala Gly Ile Tyr Asn Asp Ala Gly Lys Pro Ala Ala Val Arg Gly Leu 325 330 335 Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ala Ser Ala Pro 340 345 350 Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu 355 360 365 Ala Phe Ser Pro Leu Leu Asn Ser Ala Gly Phe Pro Ala Arg Phe Ile 370 375 380 Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp 385 390 395 400 Gly Asp Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val Arg Pro Thr 405 410

415 Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp Val Lys Pro 420 425 430 Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg Tyr Asp 435 440 445 Tyr His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala Pro Glu Ala Gly 450 455 460 Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro 465 470 475 480 Pro Phe 26516PRTTalaromyces emersonii 26Met Leu Arg Arg Ala Leu Leu Leu Ser Ser Ser Ala Ile Leu Ala Val 1 5 10 15 Lys Ala Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu 20 25 30 Thr Trp Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly 35 40 45 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly 50 55 60 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro 65 70 75 80 Asp Asp Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr 85 90 95 Glu Gly Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn 100 105 110 Phe Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp 115 120 125 Asp Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe 130 135 140 Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 145 150 155 160 Phe Val Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn 165 170 175 Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro 180 185 190 Arg Asp Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln 195 200 205 Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys 210 215 220 Cys Ala Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val 225 230 235 240 Thr Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp 245 250 255 Asp Cys Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp 260 265 270 Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe 275 280 285 Tyr Gly Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val 290 295 300 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu 305 310 315 320 Ile Lys Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn 325 330 335 Ser Asp Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys 340 345 350 Thr Ala Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His 355 360 365 Gly Gly Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu 370 375 380 Val Met Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp 385 390 395 400 Ser Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr Pro Gly Ile Ala Arg 405 410 415 Gly Thr Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln 420 425 430 Ser Pro Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile 435 440 445 Asn Ser Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Asn Arg Gly Thr 450 455 460 Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro 465 470 475 480 Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro 485 490 495 Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr 500 505 510 Ser Gln Cys Leu 515 27535PRTTrichoderma reesei 27Met Val Ser Phe Thr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly 1 5 10 15 Val Leu Ala Ala Pro Ala Ala Glu Val Glu Pro Val Ala Val Glu Lys 20 25 30 Arg Glu Ala Glu Ala Glu Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu 35 40 45 Thr His Pro Pro Leu Thr Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys 50 55 60 Thr Gln Gln Thr Gly Ser Val Val Ile Asp Ala Asn Trp Arg Trp Thr 65 70 75 80 His Ala Thr Asn Ser Ser Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser 85 90 95 Ser Thr Leu Cys Pro Asp Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu 100 105 110 Asp Gly Ala Ala Tyr Ala Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn 115 120 125 Ser Leu Ser Ile Gly Phe Val Thr Gln Ser Ala Gln Lys Asn Val Gly 130 135 140 Ala Arg Leu Tyr Leu Met Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr 145 150 155 160 Leu Leu Gly Asn Glu Phe Ser Phe Asp Val Asp Val Ser Gln Leu Pro 165 170 175 Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly 180 185 190 Gly Val Ser Lys Tyr Pro Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr 195 200 205 Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly 210 215 220 Gln Ala Asn Val Glu Gly Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr 225 230 235 240 Gly Ile Gly Gly His Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu 245 250 255 Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro His Pro Cys Thr Thr Val 260 265 270 Gly Gln Glu Ile Cys Glu Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp 275 280 285 Asn Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro 290 295 300 Tyr Arg Leu Gly Asn Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr 305 310 315 320 Leu Asp Thr Thr Lys Lys Leu Thr Val Val Thr Gln Phe Glu Thr Ser 325 330 335 Gly Ala Ile Asn Arg Tyr Tyr Val Gln Asn Gly Val Thr Phe Gln Gln 340 345 350 Pro Asn Ala Glu Leu Gly Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp 355 360 365 Tyr Cys Thr Ala Glu Glu Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp 370 375 380 Lys Gly Gly Leu Thr Gln Phe Lys Lys Ala Thr Ser Gly Gly Met Val 385 390 395 400 Leu Val Met Ser Leu Trp Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu 405 410 415 Asp Ser Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr Pro Gly Ala Val 420 425 430 Arg Gly Ser Cys Ser Thr Ser Ser Gly Val Pro Ala Gln Val Glu Ser 435 440 445 Gln Ser Pro Asn Ala Lys Val Thr Phe Ser Asn Ile Lys Phe Gly Pro 450 455 460 Ile Gly Ser Thr Gly Asn Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn 465 470 475 480 Arg Gly Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser 485 490 495 Pro Gly Pro Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr 500 505 510 Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn 515 520 525 Pro Tyr Tyr Ser Gln Cys Leu 530 535 28471PRTTrichoderma reesei 28Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala 1 5 10 15 Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly 20 25 30 Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly 35 40 45 Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly 50 55 60 Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg 65 70 75 80 Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly 85 90 95 Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr 100 105 110 Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115 120 125 Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135 140 Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu 145 150 155 160 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 165 170 175 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 180 185 190 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 195 200 205 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 210 215 220 Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 225 230 235 240 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245 250 255 Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265 270 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 275 280 285 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 290 295 300 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 305 310 315 320 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr 325 330 335 Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu 340 345 350 Tyr Ile His Ala Ile Gly Arg Leu Leu Ala Asn His Gly Trp Ser Asn 355 360 365 Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370 375 380 Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly 385 390 395 400 Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val 405 410 415 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala 420 425 430 Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala 435 440 445 Ala Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr 450 455 460 Asn Ala Asn Pro Ser Phe Leu 465 470 29532PRTChaetomium thermophilum 29Met Met Tyr Lys Lys Phe Ala Ala Leu Ala Ala Leu Val Ala Gly Ala 1 5 10 15 Ser Ala Gln Gln Ala Cys Ser Leu Thr Ala Glu Asn His Pro Ser Leu 20 25 30 Thr Trp Lys Arg Cys Thr Ser Gly Gly Ser Cys Ser Thr Val Asn Gly 35 40 45 Ala Val Thr Ile Asp Ala Asn Trp Arg Trp Thr His Thr Val Ser Gly 50 55 60 Ser Thr Asn Cys Tyr Thr Gly Asn Gln Trp Asp Thr Ser Leu Cys Thr 65 70 75 80 Asp Gly Lys Ser Cys Ala Gln Thr Cys Cys Val Asp Gly Ala Asp Tyr 85 90 95 Ser Ser Thr Tyr Gly Ile Thr Thr Ser Gly Asp Ser Leu Asn Leu Lys 100 105 110 Phe Val Thr Lys His Gln Tyr Gly Thr Asn Val Gly Ser Arg Val Tyr 115 120 125 Leu Met Glu Asn Asp Thr Lys Tyr Gln Met Phe Glu Leu Leu Gly Asn 130 135 140 Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly Leu Asn 145 150 155 160 Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Met Ser Lys 165 170 175 Tyr Ser Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185 190 Ala Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Ala Asn Val 195 200 205 Gly Asn Trp Thr Pro Ser Thr Asn Asp Ala Asn Ala Gly Phe Gly Arg 210 215 220 Tyr Gly Ser Cys Cys Ser Glu Met Asp Val Trp Glu Ala Asn Asn Met 225 230 235 240 Ala Thr Ala Phe Thr Pro His Pro Cys Thr Thr Val Gly Gln Ser Arg 245 250 255 Cys Glu Ala Asp Thr Cys Gly Gly Thr Tyr Ser Ser Asp Arg Tyr Ala 260 265 270 Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ala Tyr Arg Gln Gly 275 280 285 Asp Lys Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Asn Lys Lys 290 295 300 Met Thr Val Val Thr Gln Phe His Lys Asn Ser Ala Gly Val Leu Ser 305 310 315 320 Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Ala Asn Ala 325 330 335 Glu Ser Lys Ile Pro Gly Asn Pro Gly Asn Ser Ile Thr Gln Glu Tyr 340 345 350 Cys Asp Ala Gln Lys Val Ala Phe Ser Asn Thr Asp Asp Phe Asn Arg 355 360 365 Lys Gly Gly Met Ala Gln Met Ser Lys Ala Leu Ala Gly Pro Met Val 370 375 380 Leu Val Met Ser Val Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu 385 390 395 400 Asp Ser Thr Tyr Pro Ile Asp Gln Ala Gly Ala Pro Gly Ala Glu Arg 405 410 415 Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Ile Glu Ala Gln 420 425 430 Val Pro Asn Ser Asn Val Ile Phe Ser Asn Ile Arg Phe Gly Pro Ile 435 440 445 Gly Ser Thr Val Pro Gly Leu Asp Gly Ser Asn Pro Gly Asn Pro Thr 450 455 460 Thr Thr Val Val Pro Pro Ala Ser Thr Ser Thr Ser Arg Pro Thr Ser 465 470 475 480 Ser Thr Ser Ser Pro Val Ser Thr Pro Thr Gly Gln Pro Gly Gly Cys 485 490 495 Thr Thr Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys 500 505 510 Thr Asn Cys Val Ala Gly Thr Thr Cys Thr Gln Leu Asn Pro Trp Tyr 515 520 525 Ser Gln Cys Leu 530 30322PRTCoptotermes lacteus 30Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Glu Cys Thr Lys Gly Gly Cys Thr Asn Lys Asn Gly Tyr 20 25 30 Ile Val His Asp Lys His Val Gly Asp Ile Gln Asn Arg Asp Thr Leu 35 40 45 Asp Pro Pro Asp Leu Asp Tyr Glu Lys Asp Val Gly Val Thr Val Ser 50 55 60 Gly Gly Thr Leu Ser Gln Arg Leu Val Ser Thr Trp Asn Gly Lys Lys 65 70 75 80 Val Val Gly Ser Arg Leu Tyr Ile Val Asp Glu Ala Asp Glu Lys Tyr 85 90 95 Gln Leu Phe Thr Phe Val Gly Lys Glu Phe Thr Tyr Thr Val Asp Met 100 105 110 Ser Gln Ile Gln Cys Gly Ile Asn Ala Ala Leu Tyr Thr Val Glu Met 115 120 125 Pro Ala Ala Gly Lys Thr Pro Gly Gly Val Lys Tyr Gly Tyr Gly Tyr 130 135 140 Cys Asp Ala Asn Cys Val Asp Gly Asp Cys Cys Met Glu Phe Asp Ile 145 150 155 160 Gln Glu Ala Ser Asn Lys Ala Ile Val Tyr Thr Thr

His Ser Cys Gln 165 170 175 Ser Gln Thr Ser Gly Cys Asp Thr Ser Gly Cys Gly Tyr Asn Pro Tyr 180 185 190 Arg Asp Ser Gly Asp Lys Ala Phe Trp Gly Thr Thr Ile Asn Val Asn 195 200 205 Gln Pro Val Thr Ile Val Thr Gln Phe Ile Gly Ser Gly Ser Ser Leu 210 215 220 Thr Glu Val Lys Arg Leu Cys Val Gln Gly Gly Lys Thr Phe Pro Pro 225 230 235 240 Ala Lys Ser Leu Thr Asp Ser Tyr Cys Asn Ala Asn Asp Tyr Arg Ser 245 250 255 Leu Arg Thr Met Gly Ala Ser Met Ala Arg Gly His Val Val Val Phe 260 265 270 Ser Leu Trp Asp Ser Asn Gly Met Ser Trp Met Asp Gly Gly Asn Ala 275 280 285 Gly Pro Cys Thr Ser Tyr Asn Ile Glu Ser Leu Glu Ser Ser Gln Pro 290 295 300 Asn Leu Lys Val Thr Trp Ser Asn Val Lys Tyr Gly Glu Ile Asp Ser 305 310 315 320 Pro Tyr 31451PRTCoptotermes formosanus 31Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Lys Thr Val Leu Lys Asn Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ala Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asn Asp Lys Gly Gln Lys Gly Glu 50 55 60 Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe Gly 65 70 75 80 Phe Pro Met Ala Tyr Thr Val Thr Val Leu Ala Trp Gly Leu Val Asp 85 90 95 Tyr Glu Ser Ala Tyr Ser Thr Ala Gly Ala Leu Asp Asp Gly Arg Lys 100 105 110 Ala Leu Lys Trp Gly Thr Asp Tyr Phe Leu Lys Ala His Thr Ala Ala 115 120 125 Asn Glu Phe Tyr Gly Gln Val Gly Gln Gly Asp Val Asp His Ala Tyr 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ser Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Thr Ser Lys Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Thr Ala Ile Ala Tyr Lys Ser Ala Asp Ser Thr Tyr Ser 180 185 190 Asn Asn Leu Ile Thr His Ala Lys Gln Leu Phe Asp Phe Ala Asn Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Asp Ala Lys Asn Phe Tyr 210 215 220 Ala Ser Gly Asp Tyr Lys Asp Glu Leu Val Trp Ala Ala Ala Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Asn Thr Tyr Leu Thr Lys Ala Glu Ser Leu 245 250 255 Tyr Asn Glu Phe Gly Leu Gly Ser Trp Asn Gly Ala Phe Asn Trp Asp 260 265 270 Asn Lys Ile Ser Gly Val Gln Val Leu Leu Ala Lys Leu Thr Ser Lys 275 280 285 Gln Ala Tyr Lys Asp Lys Val Gln Gly Tyr Val Asp Tyr Leu Val Ser 290 295 300 Ser Gln Lys Lys Thr Pro Lys Gly Leu Val Tyr Ile Asp Gln Trp Gly 305 310 315 320 Thr Leu Arg His Ala Ala Asn Ser Ala Leu Ile Ala Leu Gln Ala Ala 325 330 335 Asp Leu Gly Ile Asn Ala Ala Ser Tyr Arg Gln Tyr Ala Lys Lys Gln 340 345 350 Ile Asp Tyr Ala Leu Gly Asp Gly Gly Arg Ser Tyr Val Val Gly Phe 355 360 365 Gly Thr Asn Pro Pro Val Arg Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Asp Ala Pro Ala Ala Cys Asp Trp Asn Thr Tyr Asn Ser Ala Gly Pro 385 390 395 400 Asn Ala His Val Leu Thr Gly Ala Leu Val Gly Gly Pro Asp Ser Asn 405 410 415 Asp Ser Tyr Thr Asp Ser Arg Ser Asp Tyr Ile Ser Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Gly Phe Gln Ser Ala Val Ala Gly Leu Leu Lys 435 440 445 Ala Gly Val 450 32451PRTNasutitermes takasagoensis 32Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Lys Gln Val Leu Arg Asp Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Arg Leu Pro Ala Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asn Asp Gln Gly Asp Gln Gly Gln 50 55 60 Asp Leu Thr Gly Gly Tyr Phe Asp Ala Gly Asp Phe Val Lys Phe Gly 65 70 75 80 Phe Pro Met Ala Tyr Thr Ala Thr Val Leu Ala Trp Gly Leu Ile Asp 85 90 95 Phe Glu Ala Gly Tyr Ser Ser Ala Gly Ala Leu Asp Asp Gly Arg Lys 100 105 110 Ala Val Lys Trp Ala Thr Asp Tyr Phe Ile Lys Ala His Thr Ser Gln 115 120 125 Asn Glu Phe Tyr Gly Gln Val Gly Gln Gly Asp Ala Asp His Ala Phe 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ala Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Thr Ser Arg Pro Gly Ser Asp Leu Ala Gly Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Ala Ser Ile Val Phe Arg Asn Val Asp Gly Thr Tyr Ser 180 185 190 Asn Asn Leu Leu Thr His Ala Arg Gln Leu Phe Asp Phe Ala Asn Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Asp Ala Arg Asn Phe Tyr 210 215 220 Ala Ser Ala Asp Tyr Arg Asp Glu Leu Val Trp Ala Ala Ala Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Asn Thr Tyr Leu Asn Thr Ala Glu Ser Leu 245 250 255 Tyr Asp Glu Phe Gly Leu Gln Asn Trp Gly Gly Gly Leu Asn Trp Asp 260 265 270 Ser Lys Val Ser Gly Val Gln Val Leu Leu Ala Lys Leu Thr Asn Lys 275 280 285 Gln Ala Tyr Lys Asp Thr Val Gln Ser Tyr Val Asn Tyr Leu Ile Asn 290 295 300 Asn Gln Gln Lys Thr Pro Lys Gly Leu Leu Tyr Ile Asp Met Trp Gly 305 310 315 320 Thr Leu Arg His Ala Ala Asn Ala Ala Phe Ile Met Leu Glu Ala Ala 325 330 335 Glu Leu Gly Leu Ser Ala Ser Ser Tyr Arg Gln Phe Ala Gln Thr Gln 340 345 350 Ile Asp Tyr Ala Leu Gly Asp Gly Gly Arg Ser Phe Val Cys Gly Phe 355 360 365 Gly Ser Asn Pro Pro Thr Arg Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Pro Ala Pro Ala Thr Cys Asp Trp Asn Thr Phe Asn Ser Pro Asp Pro 385 390 395 400 Asn Tyr His Val Leu Ser Gly Ala Leu Val Gly Gly Pro Asp Gln Asn 405 410 415 Asp Asn Tyr Val Asp Asp Arg Ser Asp Tyr Val His Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Gly Phe Gln Ser Ala Leu Ala Ala Leu Val Ala 435 440 445 Leu Gly Tyr 450 33451PRTCoptotermes acinaciformis 33Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Thr Thr Val Leu Lys Ser Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ala Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asp Asp Lys Gly Asn Asn Gly Glu 50 55 60 Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe Gly 65 70 75 80 Phe Pro Leu Ala Tyr Thr Ala Thr Val Leu Ala Trp Gly Leu Val Asp 85 90 95 Tyr Glu Ala Gly Tyr Ser Ser Ala Gly Ala Thr Asp Asp Gly Arg Lys 100 105 110 Ala Val Lys Trp Ala Thr Asp Tyr Leu Leu Lys Ala His Thr Ala Ala 115 120 125 Thr Glu Leu Tyr Gly Gln Val Gly Asp Gly Asp Ala Asp His Ala Tyr 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ala Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Ala Ser Arg Pro Gly Ser Asp Leu Ala Gly Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Ala Ser Ile Val Phe Lys Gly Val Asp Ser Ser Tyr Ser 180 185 190 Asp Asn Leu Leu Ala His Ala Lys Gln Leu Phe Asp Phe Ala Asp Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Gln Ala Ser Asn Phe Tyr 210 215 220 Ala Ser Gly Asp Tyr Lys Asp Glu Leu Val Trp Ala Ala Thr Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Asn Thr Tyr Leu Thr Lys Ala Glu Ser Leu 245 250 255 Tyr Asn Glu Phe Gly Leu Gly Asn Trp Asn Gly Ala Phe Asn Trp Asp 260 265 270 Asn Lys Val Ser Gly Val Gln Val Leu Leu Ala Lys Leu Thr Ser Lys 275 280 285 Gln Ala Tyr Lys Asp Thr Val Gln Gly Tyr Val Asp Tyr Leu Ile Asn 290 295 300 Asn Gln Gln Lys Thr Pro Lys Gly Leu Leu Tyr Ile Asp Gln Trp Gly 305 310 315 320 Thr Leu Arg His Ala Ala Asn Ala Ala Leu Ile Ile Leu Gln Ala Ala 325 330 335 Asp Leu Gly Ile Ser Ala Asp Ser Tyr Arg Gln Phe Ala Lys Lys Gln 340 345 350 Ile Asp Tyr Ala Leu Gly Asp Gly Gly Arg Ser Tyr Val Val Gly Phe 355 360 365 Gly Asp Asn Pro Pro Thr His Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Asp Ala Pro Ala Val Cys Asp Trp Asn Thr Phe Asn Ser Pro Asp Pro 385 390 395 400 Asn Phe His Val Leu Thr Gly Ala Leu Val Gly Gly Pro Asp Gln Asn 405 410 415 Asp Asn Tyr Val Asp Asp Arg Ser Asp Tyr Val Ser Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Gly Phe Gln Ser Ala Val Ala Ala Leu Val Thr 435 440 445 Leu Gly Val 450 34451PRTMastotermes darwinensis 34Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Asn Asp Val Leu Thr Lys Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ser Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asn Asp Lys Gly Gln Asn Gly Glu 50 55 60 Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Tyr Val Lys Phe Gly 65 70 75 80 Phe Pro Met Ala Tyr Thr Ala Thr Val Leu Ala Trp Gly Leu Val Asp 85 90 95 His Pro Ala Gly Tyr Ser Ser Ala Gly Val Leu Asp Asp Gly Arg Lys 100 105 110 Ala Val Lys Trp Val Thr Asp Tyr Leu Ile Lys Ala His Val Ser Lys 115 120 125 Asn Glu Leu Tyr Gly Gln Val Gly Asp Gly Asp Ala Asp His Ala Tyr 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ala Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Thr Ser Arg Pro Gly Ser Asp Leu Ala Gly Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Ala Ser Ile Val Phe Lys Ser Thr Asp Ser Asn Tyr Ala 180 185 190 Asn Thr Leu Leu Thr His Ala Lys Gln Leu Phe Asp Phe Ala Asn Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Gln Ala Ser Asn Phe Tyr 210 215 220 Ser Ser Ser Asp Tyr Lys Asp Glu Leu Val Trp Ala Ala Val Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Gln Thr Tyr Leu Thr Thr Ala Glu Lys Leu 245 250 255 Tyr Ser Asp Leu Gly Leu Gln Ser Trp Asn Gly Gly Phe Thr Trp Asp 260 265 270 Thr Lys Ile Ser Gly Val Glu Val Leu Leu Ala Lys Ile Thr Gly Lys 275 280 285 Gln Ala Tyr Lys Asp Lys Val Lys Gly Tyr Cys Asp Tyr Ile Ser Gly 290 295 300 Ser Gln Gln Lys Thr Pro Lys Gly Leu Val Tyr Ile Asp Lys Trp Gly 305 310 315 320 Ser Leu Arg Met Ala Ala Asn Ala Ala Tyr Ile Cys Ala Val Ala Ala 325 330 335 Asp Val Gly Ile Ser Ser Thr Ala Tyr Arg Gln Phe Ala Lys Thr Gln 340 345 350 Ile Asn Tyr Ile Leu Gly Asp Ala Gly Arg Ser Phe Val Val Gly Tyr 355 360 365 Gly Asn Asn Pro Pro Thr His Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Asp Ala Pro Ala Thr Cys Asp Trp Asn Asn Tyr Asn Ser Ala Asn Pro 385 390 395 400 Asn Pro His Val Leu Tyr Gly Ala Leu Val Gly Gly Pro Asp Ser Asn 405 410 415 Asp Asn Tyr Gln Asp Leu Arg Ser Asp Tyr Val Ala Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Ala Phe Gln Ser Leu Leu Ala Leu Ile Val Asp 435 440 445 Leu Gly Leu 450 35451PRTNasutitermes walkeri 35Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Lys Gln Val Leu Arg Asp Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Arg Leu Pro Ala Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asn Asp Gln Gly Glu Gln Gly Gln 50 55 60 Asp Leu Thr Gly Gly Tyr Phe Asp Ala Gly Asp Phe Val Lys Phe Gly 65 70 75 80 Phe Pro Met Ala Tyr Thr Ala Thr Val Leu Ala Trp Gly Leu Ile Asp 85 90 95 Phe Glu Ala Gly Tyr Ser Ser Ala Gly Ala Leu Asp Asp Gly Arg Lys 100 105 110 Ala Val Lys Trp Ala Thr Asp Tyr Phe Ile Lys Ala His Thr Ser Gln 115 120 125 Asn Glu Phe Tyr Gly Gln Val Gly Gln Gly Asp Val Asp His Ala Tyr 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ala Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Thr Ser Arg Pro Gly Ser Asp Leu Ala Gly Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Ala Ser Ile Val Phe Lys Asn Val Asp Gly Thr Tyr Ser 180 185 190 Asn Asn Leu Leu Thr His Ala Arg Gln Leu Phe Asp Phe Ala Asn Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Asp Ala Arg Asn Phe Tyr 210 215 220 Ala Ser Ala Asp Tyr Arg Asp Glu Leu Val Trp Ala Ala Ala Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Asn Ser Tyr Leu Asn Thr Ala Glu Ser Leu 245 250 255 Tyr Asn Glu Phe Gly Leu Gln Asn Trp Gly Gly Gly Leu Asn Trp Asp 260 265 270 Ser Lys Val Ser Gly Val Gln Val Leu Leu Ala Lys Leu Thr Asn Lys 275 280 285 Gln Glu Tyr Lys Asp Thr Ile Gln Ser Tyr Val Asn Tyr Leu Ile Asn 290 295 300 Asn Gln Gln Lys Thr Pro Lys Gly Leu Leu Tyr Ile Asp Met Trp Gly 305 310 315 320 Thr Leu Arg His

Ala Ala Asn Ala Ala Phe Ile Met Leu Glu Ala Ala 325 330 335 Asp Leu Gly Leu Ser Ala Ser Ser Tyr Arg Gln Phe Ala Gln Thr Gln 340 345 350 Ile Asp Tyr Ala Leu Gly Asp Gly Gly Arg Ser Phe Val Cys Gly Phe 355 360 365 Gly Ser Asn Pro Pro Thr Arg Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Pro Ala Pro Ala Thr Cys Asp Trp Asn Thr Phe Asn Ser Pro Asp Pro 385 390 395 400 Asn Tyr Asn Val Leu Ser Gly Ala Leu Val Gly Gly Pro Asp Gln Asn 405 410 415 Asp Asn Tyr Val Asp Asp Arg Ser Asp Tyr Val His Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Gly Phe Gln Ser Ala Leu Ala Ala Leu Val Ala 435 440 445 Leu Gly Tyr 450 36451PRTReticulitermes speratus 36Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Tyr Asp Tyr Lys Thr Val Leu Ser Asn Ser Leu Leu 20 25 30 Phe Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ser Asp Gln Lys Val 35 40 45 Thr Trp Arg Lys Asp Ser Ala Leu Asn Asp Lys Gly Gln Lys Gly Glu 50 55 60 Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe Gly 65 70 75 80 Phe Pro Met Ala Tyr Thr Val Thr Val Leu Ala Trp Gly Val Ile Asp 85 90 95 Tyr Glu Ser Ala Tyr Ser Ala Ala Gly Ala Leu Asp Ser Gly Arg Lys 100 105 110 Ala Leu Lys Tyr Gly Thr Asp Tyr Phe Leu Lys Ala His Thr Ala Ala 115 120 125 Asn Glu Phe Tyr Gly Gln Val Gly Gln Gly Asp Val Asp His Ala Tyr 130 135 140 Trp Gly Arg Pro Glu Asp Met Thr Met Ser Arg Pro Ala Tyr Lys Ile 145 150 155 160 Asp Thr Ser Lys Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala Ala 165 170 175 Leu Ala Ala Thr Ala Ile Ala Tyr Lys Ser Ala Asp Ala Thr Tyr Ser 180 185 190 Asn Asn Leu Ile Thr His Ala Lys Gln Leu Phe Asp Phe Ala Asn Asn 195 200 205 Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Thr Asp Ala Lys Asn Phe Tyr 210 215 220 Ala Ser Gly Asp Tyr Lys Asp Glu Leu Val Trp Ala Ala Ala Trp Leu 225 230 235 240 Tyr Arg Ala Thr Asn Asp Asn Thr Tyr Leu Thr Lys Ala Glu Ser Leu 245 250 255 Tyr Asn Glu Phe Gly Leu Gly Asn Phe Asn Gly Ala Phe Asn Trp Asp 260 265 270 Asn Lys Val Ser Gly Val Gln Val Leu Leu Ala Lys Leu Thr Ser Lys 275 280 285 Gln Val Tyr Lys Asp Lys Val Gln Ser Tyr Val Asp Tyr Leu Ile Ser 290 295 300 Ser Gln Lys Lys Thr Pro Lys Gly Leu Val Tyr Ile Asp Gln Trp Gly 305 310 315 320 Thr Leu Arg His Ala Ala Asn Ser Ala Leu Ile Ala Leu Gln Ala Ala 325 330 335 Asp Leu Gly Ile Asn Ala Ala Thr Tyr Arg Ala Tyr Ala Lys Lys Gln 340 345 350 Ile Asp Tyr Ala Leu Gly Asp Gly Gly Arg Ser Tyr Val Ile Gly Phe 355 360 365 Gly Thr Asn Pro Pro Val Arg Pro His His Arg Ser Ser Ser Cys Pro 370 375 380 Asp Ala Pro Ala Val Cys Asp Trp Asn Thr Tyr Asn Ser Ala Gly Pro 385 390 395 400 Asn Ala His Val Leu Thr Gly Ala Leu Val Gly Gly Pro Asp Ser Asn 405 410 415 Asp Ser Tyr Thr Asp Ala Arg Ser Asp Tyr Ile Ser Asn Glu Val Ala 420 425 430 Thr Asp Tyr Asn Ala Gly Phe Gln Ser Ala Val Ala Gly Leu Leu Lys 435 440 445 Ala Gly Val 450 37408PRTNeosartorya fischeri 37Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Gln Gln Ile Gly Ser Ile Ala Glu Asn His Pro Glu Leu 20 25 30 Thr Thr Tyr Arg Cys Ser Ser Gln Ala Gly Cys Val Ala Gln Ser Thr 35 40 45 Ser Val Val Leu Asp Ile Asn Ala His Trp Ile His Gln Asn Gly Ala 50 55 60 Gln Thr Ser Cys Thr Thr Ser Ser Gly Leu Asp Pro Ser Leu Cys Pro 65 70 75 80 Asp Lys Val Thr Cys Ser Gln Asn Cys Val Val Glu Gly Ile Thr Asp 85 90 95 Tyr Ser Ser Phe Gly Val Gln Asn Ser Gly Asp Ala Met Thr Leu Arg 100 105 110 Gln Tyr Gln Val Gln Asn Gly Gln Ile Lys Thr Leu Arg Pro Arg Val 115 120 125 Tyr Leu Leu Ala Glu Asp Gly Ile Asn Tyr Ser Lys Leu Gln Leu Leu 130 135 140 Asn Gln Glu Phe Thr Phe Asp Val Asp Ala Ser Lys Leu Pro Cys Gly 145 150 155 160 Met Asn Gly Ala Leu Tyr Leu Ser Glu Met Asp Ala Ser Gly Gly Arg 165 170 175 Ser Ala Leu Asn Pro Ala Gly Ala Thr Tyr Gly Thr Gly Tyr Cys Asp 180 185 190 Ala Gln Cys Phe Asn Pro Gly Pro Trp Ile Asn Gly Glu Ala Asn Thr 195 200 205 Ala Gly Ala Gly Ala Cys Cys Gln Glu Met Asp Leu Trp Glu Ala Asn 210 215 220 Ser Arg Ser Thr Ile Phe Ser Pro His Pro Cys Thr Thr Ala Gly Leu 225 230 235 240 Tyr Ala Cys Thr Gly Ala Glu Cys Tyr Ser Ile Cys Asp Gly Tyr Gly 245 250 255 Cys Thr Tyr Asn Pro Tyr Glu Leu Gly Ala Lys Asp Tyr Tyr Gly Tyr 260 265 270 Gly Leu Thr Ile Asp Thr Ala Lys Pro Ile Thr Val Val Thr Gln Phe 275 280 285 Met Thr Ala Asp Asn Thr Ala Thr Gly Thr Leu Ala Glu Ile Arg Arg 290 295 300 Leu Tyr Val Gln Asp Gly Lys Val Ile Gly Asn Thr Ala Val Ala Met 305 310 315 320 Thr Glu Ala Phe Cys Ser Ser Ser Arg Thr Phe Glu Glu Leu Gly Gly 325 330 335 Leu Gln Arg Met Gly Glu Ala Leu Gly Arg Gly Met Val Pro Val Phe 340 345 350 Ser Ile Trp Asp Asp Pro Gly Leu Trp Met His Trp Leu Asp Ser Asp 355 360 365 Gly Ala Gly Pro Cys Gly Asn Thr Glu Gly Asp Pro Ala Phe Ile Gln 370 375 380 Ala Asn Tyr Pro Asn Thr Ala Val Thr Phe Ser Lys Val Arg Trp Gly 385 390 395 400 Asp Ile Gly Ser Thr Tyr Ser Ser 405 38304PRTReticulitermes flavipes 38Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Gln Trp Met Gln Ile Gly Gly Lys Gln Lys Tyr Pro Ala 20 25 30 Phe Lys Pro Gly Ala Lys Tyr Gly Arg Gly Tyr Cys Asp Gly Gln Cys 35 40 45 Pro His Asp Met Lys Val Ser Ser Gly Arg Ala Asn Val Asp Gly Trp 50 55 60 Lys Pro Gln Asp Asn Asp Glu Asn Ser Gly Asn Gly Lys Leu Gly Thr 65 70 75 80 Cys Cys Trp Glu Met Asp Ile Trp Glu Gly Asn Leu Val Ser Gln Ala 85 90 95 Tyr Thr Val His Ala Gly Ser Lys Ser Gly Gln Tyr Glu Cys Thr Gly 100 105 110 Thr Gln Cys Gly Asp Thr Asp Ser Gly Glu Arg Phe Lys Gly Thr Cys 115 120 125 Asp Lys Asp Gly Cys Asp Phe Ala Ser Tyr Arg Trp Gly Ala Thr Asp 130 135 140 Tyr Tyr Gly Pro Gly Lys Thr Val Asp Thr Lys Gln Pro Met Thr Val 145 150 155 160 Val Thr Gln Phe Ile Gly Asp Pro Leu Thr Glu Ile Lys Arg Val Tyr 165 170 175 Val Gln Gly Gly Lys Val Ile Asn Asn Ser Lys Thr Ser Asn Leu Gly 180 185 190 Ser Val Tyr Asp Ser Leu Thr Glu Ala Phe Cys Asp Asp Thr Lys Gln 195 200 205 Val Thr Gly Asp Thr Asn Asp Phe Lys Ala Lys Gly Gly Met Ser Gly 210 215 220 Phe Ser Lys Asn Leu Asp Thr Pro Gln Val Leu Val Met Ser Leu Trp 225 230 235 240 Asp Asp His Thr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr 245 250 255 Asp Ser Thr Lys Pro Gly Ala Ala Arg Gly Thr Cys Ala Val Thr Ser 260 265 270 Gly Asp Pro Lys Asp Val Glu Ser Lys Gln Ala Asn Ser Gln Val Val 275 280 285 Tyr Ser Asp Ile Lys Phe Gly Pro Ile Asn Ser Thr Tyr Lys Ala Asn 290 295 300 39475PRTTrichoderma reesei 39Met Val Ser Phe Thr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly 1 5 10 15 Val Leu Ala Ala Pro Ala Ala Glu Val Glu Pro Val Ala Val Glu Lys 20 25 30 Arg Glu Ala Glu Ala Glu Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu 35 40 45 Val His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys 50 55 60 Val Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met 65 70 75 80 His Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr 85 90 95 Thr Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu 100 105 110 Gly Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu 115 120 125 Thr Met Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val 130 135 140 Ser Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr Val Met Leu 145 150 155 160 Lys Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu 165 170 175 Pro Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn 180 185 190 Gly Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly 195 200 205 Tyr Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu 210 215 220 Asn Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu 225 230 235 240 Gly Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr 245 250 255 Ala Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr 260 265 270 Lys Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe 275 280 285 Thr Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn 290 295 300 Leu Val Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro 305 310 315 320 Ser Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser 325 330 335 Ala Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met 340 345 350 Val Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp 355 360 365 Leu Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro 370 375 380 Ser Asn Ile Leu Ala Asn Asn Pro Asn Thr His Val Val Phe Ser Asn 385 390 395 400 Ile Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro 405 410 415 Pro Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser 420 425 430 Thr Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys 435 440 445 Gly Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr 450 455 460 Cys Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys 465 470 475 40896PRTSaccharomycopsis fibuligera 40Met Val Ser Phe Thr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly 1 5 10 15 Val Leu Ala Ala Pro Ala Ala Glu Val Glu Ser Val Ala Val Glu Lys 20 25 30 Arg Ser Asp Ser Arg Val Pro Ile Gln Asn Tyr Thr Gln Ser Pro Ser 35 40 45 Gln Arg Asp Glu Ser Ser Gln Trp Val Ser Pro His Tyr Tyr Pro Thr 50 55 60 Pro Gln Gly Gly Arg Leu Gln Asp Val Trp Gln Glu Ala Tyr Ala Arg 65 70 75 80 Ala Lys Ala Ile Val Gly Gln Met Thr Ile Val Glu Lys Val Asn Leu 85 90 95 Thr Thr Gly Thr Gly Trp Gln Leu Asp Pro Cys Val Gly Asn Thr Gly 100 105 110 Ser Val Pro Arg Phe Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro 115 120 125 Leu Gly Val Arg Phe Ala Asp Phe Val Thr Gly Tyr Pro Ser Gly Leu 130 135 140 Ala Thr Gly Ala Thr Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln 145 150 155 160 Ala Leu Gly His Glu Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly 165 170 175 Pro Ala Val Gly Pro Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe 180 185 190 Glu Ala Phe Gly Ser Asp Pro Tyr Leu Gln Gly Thr Ala Ala Ala Ala 195 200 205 Thr Ile Lys Gly Leu Gln Glu Asn Asn Val Met Ala Cys Val Lys His 210 215 220 Phe Ile Gly Asn Glu Gln Glu Lys Tyr Arg Gln Pro Asp Asp Ile Asn 225 230 235 240 Pro Ala Thr Asn Gln Thr Thr Lys Glu Ala Ile Ser Ala Asn Ile Pro 245 250 255 Asp Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ser Val 260 265 270 Arg Ala Gly Val Gly Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn 275 280 285 Thr Tyr Ala Cys Glu Asn Ser Tyr Met Met Asn His Leu Leu Lys Glu 290 295 300 Glu Leu Gly Phe Gln Gly Phe Val Val Ser Asp Trp Gly Ala Gln Leu 305 310 315 320 Ser Gly Val Tyr Ser Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly 325 330 335 Glu Val Tyr Gly Gly Trp Asn Thr Gly Thr Ser Phe Trp Gly Gln Asn 340 345 350 Leu Thr Lys Ala Ile Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp 355 360 365 Asp Met Ala Thr Arg Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe 370 375 380 Pro Thr Glu Asp His Leu Pro Asn Phe Ser Ser Trp Thr Thr Lys Glu 385 390 395 400 Tyr Gly Asn Lys Tyr Tyr Ala Asp Asn Thr Thr Glu Ile Val Lys Val 405 410 415 Asn Tyr His Val Asp Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu 420 425 430 Lys Val Ala Glu Glu Ser Ile Val Leu Leu Lys Asn Glu Asn Asn Thr 435 440 445 Leu Pro Ile Ser Pro Glu Lys Ala Lys Arg Leu Leu Leu Ser Gly Ile 450 455 460 Ala Ala Gly Pro Asp Pro Ile Gly Tyr Gln Cys Glu Asp Gln Ser Cys 465 470 475 480 Thr Asn Gly Ala Leu Phe Gln Gly Trp Gly Ser Gly Ser Val Gly Ser 485 490 495 Pro Lys Tyr Gln Val Thr Pro Phe Glu Glu Ile Ser Tyr Leu Ala Arg 500 505 510 Lys Asn Lys Met Gln Phe Asp Tyr Ile Arg Glu Ser Tyr Asp

Leu Ala 515 520 525 Gln Val Thr Lys Val Ala Ser Asp Ala His Leu Ser Ile Val Val Val 530 535 540 Ser Ala Ala Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Gln Gly 545 550 555 560 Asp Arg Arg Asn Leu Thr Leu Trp Asn Asn Gly Asp Lys Leu Ile Glu 565 570 575 Thr Val Ala Glu Asn Cys Ala Asn Thr Val Val Val Val Thr Ser Thr 580 585 590 Gly Gln Ile Asn Phe Glu Gly Phe Ala Asp His Pro Asn Val Thr Ala 595 600 605 Ile Val Trp Ala Gly Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala 610 615 620 Asn Ile Leu Phe Gly Lys Ala Asn Pro Ser Gly His Leu Pro Phe Thr 625 630 635 640 Ile Ala Lys Thr Asp Asp Asp Tyr Ile Pro Ile Glu Thr Tyr Ser Pro 645 650 655 Ser Ser Gly Glu Pro Glu Asp Asn His Leu Val Glu Asn Asp Leu Leu 660 665 670 Val Asp Tyr Arg Tyr Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala 675 680 685 Phe Gly Tyr Gly Leu Ser Tyr Asn Glu Tyr Glu Val Ser Asn Ala Lys 690 695 700 Val Ser Ala Ala Lys Lys Val Asp Glu Glu Leu Pro Glu Pro Ala Thr 705 710 715 720 Tyr Leu Ser Glu Phe Ser Tyr Gln Asn Ala Lys Asp Ser Lys Asn Pro 725 730 735 Ser Asp Ala Phe Ala Pro Thr Asp Leu Asn Arg Val Asn Glu Tyr Leu 740 745 750 Tyr Pro Tyr Leu Asp Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu 755 760 765 Tyr Pro Asp Gly Tyr Ser Thr Glu Gln Arg Thr Thr Pro Ile Gln Pro 770 775 780 Gly Gly Gly Leu Gly Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Lys 785 790 795 800 Val Glu Val Asp Val Gln Asn Leu Gly Asn Ser Thr Asp Lys Phe Val 805 810 815 Pro Gln Leu Tyr Leu Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro 820 825 830 Ile Gln Leu Arg Gly Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys 835 840 845 Lys Thr Val Glu Phe Glu Leu Leu Arg Arg Asp Leu Ser Val Trp Asp 850 855 860 Thr Thr Arg Gln Ser Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu 865 870 875 880 Ile Gly Val Ala Val Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile 885 890 895 41144DNAArtificial SequenceDescription of Artificial Sequence Synthetic flexible linker polynucleotide 41ggaggaggtg gttcaggagg tggtgggtct gcttggcatc cacaatttgg aggaggcggt 60ggtgaaaatc tgtatttcca gggaggcgga ggtgattaca aggatgacga caaaggaggt 120ggtggatcag gaggtggtgg ctcc 14442117DNAArtificial SequenceDescription of Artificial Sequence Synthetic flexible linker polynucleotide 42ggtggcggtg gatctggagg aggcggttct tggtctcacc cacaatttga aaagggtgga 60gaaaacttgt actttcaagg cggtggtgga ggttctggcg gaggtggctc cggctca 11743455PRTArtificial SequenceDescription of Artificial Sequence Synthetic CBH1 consensus polypeptide 43Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Gln Gln Ala Gly Thr Leu Thr Ala Glu Thr His Pro Ser 20 25 30 Leu Thr Trp Gln Lys Cys Thr Ser Gly Gly Ser Cys Thr Thr Val Asn 35 40 45 Gly Ser Val Val Ile Asp Ala Asn Trp Arg Trp Val His Ala Thr Ser 50 55 60 Gly Ser Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr Thr Leu Cys 65 70 75 80 Pro Asp Asp Val Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp 85 90 95 Tyr Ser Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Arg Leu 100 105 110 Asn Phe Val Thr Gln Gly Ser Gln Lys Asn Val Gly Ser Arg Leu Tyr 115 120 125 Leu Met Glu Asp Asp Thr Thr Tyr Gln Met Phe Lys Leu Leu Gly Gln 130 135 140 Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn 145 150 155 160 Gly Ala Leu Tyr Phe Val Ala Met Asp Ala Asp Gly Gly Met Ser Lys 165 170 175 Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185 190 Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val 195 200 205 Glu Gly Trp Glu Pro Ser Ser Asn Asp Ala Asn Ala Gly Ile Gly Asn 210 215 220 His Gly Ser Cys Cys Ala Glu Met Asp Ile Trp Glu Ala Asn Ser Ile 225 230 235 240 Ser Thr Ala Phe Thr Pro His Pro Cys Asp Thr Ile Gly Gln Thr Met 245 250 255 Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Ser Asp Arg Tyr Gly 260 265 270 Gly Thr Cys Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly 275 280 285 Asn Lys Thr Phe Tyr Gly Pro Gly Lys Thr Val Asp Thr Thr Lys Lys 290 295 300 Val Thr Val Val Thr Gln Phe Ile Thr Gly Ser Ser Gly Thr Leu Ser 305 310 315 320 Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Asn Ser 325 330 335 Glu Ser Thr Ile Ser Gly Val Ser Gly Asn Ser Ile Thr Thr Asp Phe 340 345 350 Cys Thr Ala Gln Lys Thr Ala Phe Gly Asp Thr Asp Asp Phe Ala Lys 355 360 365 Lys Gly Gly Leu Glu Gly Met Gly Lys Ala Leu Ala Gln Gly Met Val 370 375 380 Leu Val Met Ser Leu Trp Asp Asp His Ala Ala Asn Met Leu Trp Leu 385 390 395 400 Asp Ser Thr Tyr Pro Thr Asp Ala Thr Ser Ser Thr Pro Gly Ala Ala 405 410 415 Arg Gly Ser Cys Asp Thr Ser Ser Gly Val Pro Ala Asp Val Glu Ala 420 425 430 Asn Ser Pro Asn Ser Tyr Val Thr Phe Ser Asn Ile Lys Phe Gly Pro 435 440 445 Ile Gly Ser Thr Phe Thr Gly 450 455 441368DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon optimized CBH1 consensus polynucleotide 44atgagatttc cttcaatctt cactgctgtt ttgttcgcag cctcaagtgc tttagcacaa 60caggccggaa cattgacagc agaaactcat ccttccttaa cctggcaaaa gtgcacttct 120ggaggttcat gcactacagt gaatggatct gtcgtgatcg atgcaaactg gagatgggtt 180cacgcaactt caggttctac caactgttat accggaaaca cttgggacac cacattgtgc 240ccagatgacg tcacgtgcgc tcagaactgt gctttggatg gagctgatta cagttcaacc 300tatggtgtaa ctacatccgg aaactctttg agattaaact tcgttactca aggaagtcaa 360aagaacgttg gttctagatt gtacttaatg gaggacgata caacctatca aatgttcaaa 420ttgttaggtc aggagttcac ctttgacgta gatgtcagta acttgccatg tgggttaaac 480ggagctttat actttgtggc aatggatgct gacggtggaa tgtccaagta tccaggaaac 540aaagccggtg caaagtacgg tacaggatat tgtgattcac agtgccctag agatttgaag 600ttcattaacg gtcaagcaaa tgtggagggt tgggaaccat ctagtaacga tgccaatgcg 660ggtattggta atcatgggtc ctgttgcgct gagatggata tctgggaggc caactcaata 720tctactgcct ttacccctca cccatgcgat acaattggtc aaactatgtg cgagggtgat 780tcatgtggtg gaacctactc ctctgataga tacggaggta catgcgatcc agatggttgc 840gactttaatc catacagaat gggaaacaaa accttttacg gtcctggaaa gacagttgat 900actaccaaga aagtaacagt cgtgacccag tttatcaccg gtagttctgg aaccttatcc 960gaaatcaaaa gattctacgt tcagaacggt aaagtaattc caaacagtga atctacaatt 1020tcaggagtga gtggtaattc tattactacc gacttttgta cagctcagaa aacagcattt 1080ggtgacaccg atgactttgc taagaagggt ggattagaag gtatgggtaa agctttggcc 1140cagggaatgg tgttagttat gtctttatgg gatgatcacg ccgcaaatat gttatggttg 1200gattcaacat atccaactga tgccacaagt agtacacctg gagctgccag aggttcttgt 1260gatacatctt ccggtgttcc agccgatgta gaagcaaatt ctcctaactc ctatgttacc 1320ttctccaata taaagtttgg tccaatcggt tcaacattca ctggttaa 1368451572DNAAcremonium thermophilum 45atgtatacca aatttgctgc attggccgct ttagttgcaa cagtaagagg tcaagccgct 60tgttctctaa ccgcagaaac tcacccatct ctacaatggc agaaatgcac agcccctgga 120tcttgtacaa ctgtctccgg ccaagtcacc attgacgcta attggagatg gcttcaccaa 180actaactctt caacgaattg ttataccggt aacgaatggg atacttccat atgttcatcc 240gatacagact gcgcaacgaa atgttgttta gatggagcag actatacggg aacttatggt 300gttacagcct caggtaattc cctaaacctt aagttcgtaa ctcaaggacc atatagtaag 360aatatcggct ctagaatgta cttgatggaa agtgagagca aatatcaggg ttttacgtta 420ttgggacaag agtttacatt tgatgttgat gtgagtaact taggttgcgg cctaaacggc 480gccttgtact tcgtttctat ggatcttgat ggaggtgtat caaaatacac gaccaacaag 540gctggagcca aatatggtac gggatattgt gacagccaat gccctagaga cttaaagttc 600attaacggtc aggcaaatat tgacggctgg caaccaagca gtaacgacgc taatgccgga 660ctaggtaacc atggctcatg ttgttccgaa atggatatct gggaagccaa taaggtgtcc 720gctgcctaca ccccccatcc atgcacgaca atcggtcaga caatgtgtac cggtgatgac 780tgtggaggca catactcaag tgataggtac gccggtatat gtgatcctga cggttgcgat 840ttcaactctt atagaatggg agatacatcc ttttacggcc ccggtaaaac agttgatacg 900ggtagtaagt tcactgttgt tactcagttc ttaacaggtt cagacggcaa tcttagtgaa 960atcaaaagat tctacgttca gaatggaaaa gtcattccta attccgagag taagattgct 1020ggtgtgtctg gtaacagtat cacgaccgac ttctgtaccg cccaaaagac tgcctttgga 1080gatacgaatg ttttcgagga aaggggcggt cttgctcaaa tgggcaaggc tttggccgaa 1140ccaatggtat tagtcctatc cgtttgggat gatcatgcag tgaatatgct ttggcttgat 1200agcacctacc ctactgacag caccaagcca ggagctgcca gaggtgactg tcctatcaca 1260agtggcgttc cagcagatgt agagagccaa gctccaaact ccaatgtgat ctattctaac 1320atcagatttg gccccattaa tagtacctat acaggaacgc cctctggtgg taaccctcca 1380ggcggaggca ccacaactac cacgaccaca acgacttcaa agccttctgg ccctacgaca 1440actaccaatc cttccggacc acagcaaact cactggggtc agtgtggagg ccaaggatgg 1500acgggtccta ccgtgtgtca atcaccttac acatgcaaat acagtaatga ctggtactct 1560cagtgtttat aa 157246523PRTAcremonium thermophilum 46Met Tyr Thr Lys Phe Ala Ala Leu Ala Ala Leu Val Ala Thr Val Arg 1 5 10 15 Gly Gln Ala Ala Cys Ser Leu Thr Ala Glu Thr His Pro Ser Leu Gln 20 25 30 Trp Gln Lys Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Ser Gly Gln 35 40 45 Val Thr Ile Asp Ala Asn Trp Arg Trp Leu His Gln Thr Asn Ser Ser 50 55 60 Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser Ile Cys Ser Ser 65 70 75 80 Asp Thr Asp Cys Ala Thr Lys Cys Cys Leu Asp Gly Ala Asp Tyr Thr 85 90 95 Gly Thr Tyr Gly Val Thr Ala Ser Gly Asn Ser Leu Asn Leu Lys Phe 100 105 110 Val Thr Gln Gly Pro Tyr Ser Lys Asn Ile Gly Ser Arg Met Tyr Leu 115 120 125 Met Glu Ser Glu Ser Lys Tyr Gln Gly Phe Thr Leu Leu Gly Gln Glu 130 135 140 Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly Leu Asn Gly 145 150 155 160 Ala Leu Tyr Phe Val Ser Met Asp Leu Asp Gly Gly Val Ser Lys Tyr 165 170 175 Thr Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser 180 185 190 Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Ile Asp 195 200 205 Gly Trp Gln Pro Ser Ser Asn Asp Ala Asn Ala Gly Leu Gly Asn His 210 215 220 Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Lys Val Ser 225 230 235 240 Ala Ala Tyr Thr Pro His Pro Cys Thr Thr Ile Gly Gln Thr Met Cys 245 250 255 Thr Gly Asp Asp Cys Gly Gly Thr Tyr Ser Ser Asp Arg Tyr Ala Gly 260 265 270 Ile Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Met Gly Asp 275 280 285 Thr Ser Phe Tyr Gly Pro Gly Lys Thr Val Asp Thr Gly Ser Lys Phe 290 295 300 Thr Val Val Thr Gln Phe Leu Thr Gly Ser Asp Gly Asn Leu Ser Glu 305 310 315 320 Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu 325 330 335 Ser Lys Ile Ala Gly Val Ser Gly Asn Ser Ile Thr Thr Asp Phe Cys 340 345 350 Thr Ala Gln Lys Thr Ala Phe Gly Asp Thr Asn Val Phe Glu Glu Arg 355 360 365 Gly Gly Leu Ala Gln Met Gly Lys Ala Leu Ala Glu Pro Met Val Leu 370 375 380 Val Leu Ser Val Trp Asp Asp His Ala Val Asn Met Leu Trp Leu Asp 385 390 395 400 Ser Thr Tyr Pro Thr Asp Ser Thr Lys Pro Gly Ala Ala Arg Gly Asp 405 410 415 Cys Pro Ile Thr Ser Gly Val Pro Ala Asp Val Glu Ser Gln Ala Pro 420 425 430 Asn Ser Asn Val Ile Tyr Ser Asn Ile Arg Phe Gly Pro Ile Asn Ser 435 440 445 Thr Tyr Thr Gly Thr Pro Ser Gly Gly Asn Pro Pro Gly Gly Gly Thr 450 455 460 Thr Thr Thr Thr Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr 465 470 475 480 Thr Thr Asn Pro Ser Gly Pro Gln Gln Thr His Trp Gly Gln Cys Gly 485 490 495 Gly Gln Gly Trp Thr Gly Pro Thr Val Cys Gln Ser Pro Tyr Thr Cys 500 505 510 Lys Tyr Ser Asn Asp Trp Tyr Ser Gln Cys Leu 515 520 471586DNAAspergillus kawachii 47ttaattaaaa tgagaatttc taacttgatt gttgctgctt ctgctgctac tatggtttct 60gctttgccat ctagacaaat gaaaaagagg gattctggtt ttaaatgggt tggtacttct 120gaatctggtg ctgaatttgg ttctgcttta ccaggtactt tgggtactga ttatacttgg 180ccagaaactt ctaaaattca agttttgaga aacaagggta tgaacatttt tagaatacca 240ttcttgatgg aaagattaac tccagatggt ttgactggtt cttttgcttc tacttacttg 300tctgatttga agtcaactgt tgaatttgtt actaattctg gtgcttatgc tgttttagat 360ccacataatt acggtagatt cgatggttct attattgaat ctacttctga ttttaagact 420tggtggaaaa atgttgctac tgaatttgct gataacgata aggttatttt cgatacaaac 480aacgaatatc atgatatgga acaatctttg gttttgaatt tgaaccaagc tgctattaat 540ggtattagag ctgctggtgc tactactcaa tacattttcg ttgaaggtaa tgcttatact 600ggtgcttggg attggactac ttacaatgat gatttgtctg gtttaactga ttctgaagat 660aagataatat acgaaatgca tcaatacttg gattctgatt cttctggtac atctgaaact 720tgtgtttctt ctactattgg taaagaaaga attgaaaagg ctactgaatg gttgaaaact 780aacaacaagc aaggtattat tggtgaattt gcaggtggtg ttaattctgt ttgtgaagag 840gctgttgaag gaatgttggc ttatatgtct gaaaattctg atgtttgggt tggtgcttct 900tggtggtctg ctggtccatg gtggggtact tacatgtatt ctttggaacc aactgatggt 960actgcttatt ctacttattt gccaattttg gaaaaatact tcccatctgg tgatgcttca 1020tcatcttcat ctgcttcagc ttcagttgca gccgctactt ctgctgtttc tactactact 1080acagctgcat ttgaacaaac tactactcca gctactcaag ttgaaattgc ttcttcttca 1140tcttcatcat cagctgttgc tgcttcacaa actactttgt ctaaggttaa gtctaaatct 1200aaatctccat gtaaattgtc atctgctact tcatctgctg tttcatcagc tgctgcagtt 1260actacacctg cagttgcagc tacaactcca gctgctgctc caacttcttc ttctgttgct 1320tttgctacta cttctgttta cgttccaact actactgctg ctgcaccatc tcaagtttca 1380tcttcagctg cagcttcatc ttcaggtgtt gttggtgttt ctgatccaca aggtccatct 1440gctactaatt ctgctggtga agttaatcaa tattaccaat gtggtggtat taattggact 1500ggtccaactg tttgtgcttc tccatatact tgtaaggttc aaaacgatta ctactatcaa 1560tgtgttgctg aattataagg cgcgcc 1586481280DNAHeterodera schachtii 48ttaattaaaa tgcattgggc tgatgttgct tgttctagac caccatggcc aagagattct 60gttaaagctt tgaagtgtaa ttggaacgct aatgttatta gaggtgctat gggtgttgat 120gaaggtggtt atttgtctga tgctaatact gcttacaatt tgatggttgc tgttattgaa 180gctgctattt ctaatggtat ctacgttatt gttgattggc atgctcataa tgctcatcca 240gatgaagctg ttaaattctt tactagaatt gctcaagctt atggttctta cttgcatatt 300ttgtacgaag atttcaatga accattggat gtttcttgga ctgatgtttt ggttccatac 360cataaaaaag ttattgctgc cattagagct attgataaga agaacgttat tatcttgggt 420actccaaaat ggtcacaaga tgttgatgtt gcttctcaaa atccaattaa ggattaccaa 480aacttgatgt acactttgca tttttacgct tcatctcatt ttacatctga tttgggtgct 540aaattgaaaa ctgctgttaa caatggtttg ccagtttttg ttactgaata tggtacttgt 600gaagcttctg gtaatggtaa tttgaatact gattctatgt catcttggtg gactttgttg 660gattctttga aaatttctta cgctaattgg gctatttctg ataaatctga agcttgttct 720gctttgtctc caggtactac tgctgttaat gttggtgttt cttctagatg gacttcttct

780ggtaatatgg ttgcttctta ctacaaaaaa aagtccactg gtatttcttg ttctggtagt 840tcttcaggtt cttcaagtgg ttcatctagt ggttcttccg gtacatcttc tggttctagt 900ggttcatcta gtggtagttc ttccggtagt tctagtggta gttctggttc aagttctggt 960tcctcctctg gttctggttc tgcatctatt tctgttgttc catctaatac ttggaatggt 1020ggtggtagag ttaattttga aattaagaac actggttctg ttccattgtg tggtgttgtt 1080ttttctgttt ctttgccatc tggtactact ttgggtggtt cttggaatat ggaatctgct 1140ggttctggtc aatattcttt accatcttgg gttagaattg aagctggtaa atcttctaaa 1200gatgctggtt tgacttttaa tggtaaagat aagccaactg ctaaaattgt taccaccaag 1260aagtgcttat aaggcgcgcc 1280491277DNAHypocrea jecorina 49ttaattaaaa tgaacaagtc tgttgctcca ttgttgttgg ctgcttctat tttgtatggt 60ggtgctgttg ctcaacaaac tgtttggggt caatgtggtg gtattggttg gtctggtcca 120actaattgtg ctccaggttc tgcttgttct actttgaatc catattatgc tcaatgtatt 180ccaggtgcta ctactattac tacttctact agaccaccat ctggtccaac aactactact 240agagctactt ctacatcttc ttctactcca ccaacttcat ctggtgttag atttgctggt 300gttaacattg ctggttttga ttttggttgt actactgatg gtacttgtgt tacttctaaa 360gtttacccac cattgaaaaa tttcactggt tctaacaatt atccagatgg tattggtcaa 420atgcaacatt ttgttaacga agatggtatg actattttta gattgccagt tggttggcaa 480tatttggtta acaacaattt gggtggtaat ttggattcta cttctatttc taagtacgat 540caattggttc aaggttgttt gtctttgggt gcttactgta ttgttgatat tcataattat 600gctagatgga atggtggtat tattggtcaa ggtggtccaa caaatgctca atttacttct 660ttgtggtcac aattggcttc aaaatatgct tctcaatcta gagtttggtt tggtattatg 720aatgaaccac atgatgttaa cattaatact tgggctgcta ctgttcaaga agttgttact 780gctattagaa atgctggtgc tacttctcaa ttcatttctt tgccaggtaa tgattggcaa 840tctgctggtg cttttatttc tgatggttct gctgctgctt tgtctcaagt tactaatcca 900gatggttcta ctactaattt gatcttcgat gttcataagt acttggattc tgataattct 960ggtactcatg ctgaatgtac tacaaacaat attgatggtg ctttttctcc attggctact 1020tggttgagac aaaacaatag acaagctatt ttgactgaaa ctggtggtgg taatgttcaa 1080tcttgtatcc aagatatgtg ccaacaaatt caatacttga accaaaattc tgatgtttat 1140ttgggttacg ttggttgggg tgctggttct tttgattcta cttacgtttt aactgaaact 1200ccaacttctt ctggtaattc ttggactgat acttctttgg tttcttcatg tttggctaga 1260aagttataag gcgcgcc 1277501436DNAOrpinomyces sp. 50ttaattaaaa tgaagttctt gaactctttg tctttgttgg gtttggttat tgctggttgt 60gaagctatga gaaacatttc ttctaaagaa ttggttaaag aattgactat tggttggtct 120ttgggtaata ctttggatgc ttcttgtgtt gaaactttga actactctaa agatcaaact 180gcttctgaaa cttgttgggg taatgttaaa actactcaag aattgtacta caaattgtct 240gatttgggtt tcaatacttt cagaatacca actacttggt ctggtcattt tggtgatgct 300ccagattaca aaatttctga tgtttggatg aaaagagttc acgaagttgt tgattatgct 360ttgaatactg gtggttacgc tattttgaac attcatcatg aaacttggaa ttacgctttt 420caaaagaatt tggaatctgc taaaaagatt ttggttgcta tttggaaaca aattgctgct 480gaatttggtg attacgatga acatttgatt tttgaaggta tgaatgaacc aagaaaagtt 540ggtgatccag ctgaatggac tggtggtgat caagaaggtt ggaattttgt taatgaaatg 600aacgctttgt tcgttaaaac tattagagct actggtggta acaatgctaa tagacatttg 660atgattccaa cttatgctgc ttctgttaat gatggttcta ttaacaattt taagtaccca 720aatggtgatg ataaagttat tgtttctttg cattcttact ctccatacaa ttttgctttg 780aacaatggtc caggtgctat ttctaatttc tacgatggta acgaaattga ttgggttatg 840aacactatta actcttcatt catttctaag ggtattccag ttattattgg tgaatttgtt 900gctatgaaca gagataatga agatgataga gaaagatggc aagaatacta cattaaaaag 960gctactgctt tgggtattcc atgtgttatt tgggataatg gttattttga aggtgaaggt 1020gaaagatttg gtattattga tagaaagtct ttgaacgtta ttttcccaaa gttgattaat 1080ggtttgatga aaggtttggg tgatgaaaaa ccaaaaacta ctattagaag aactactact 1140actacagttc aagttcaacc aactattaac aacgaatgtt tctctactag attgggttat 1200tcttgttgta atggtttcga tgttttgtac actgataatg atggtcaatg gggtgttgaa 1260aatggtaatt ggtgtggtat taaatcttct tgtggtaaca atcaaagaca atgttggtct 1320gaaagattag gttatccatg ttgtcaatac actactaatg ctgaatatac agacaacgac 1380ggtagatggg gtgtagaaaa cggtaactgg tgcggaatat acttgtaagg cgcgcc 1436511220DNAIrpex lacteus 51ttaattaaaa tgaagtcttt gttgttgtct gctgctgcta ctttggcttt atctactcca 60gctttttctg tttctgtttg gggtcaatgt ggtggtattg gttttactgg ttctactact 120tgtgatgctg gtacttcttg tgttcatttg aacgattact actttcaatg tcaaccaggt 180gctgctactt ctactgttca accaactact actgcttctt ctacttcttc tgctgcagct 240ccatcttctt caggtaatgc tgtttgttct ggtactagaa acaagtttaa gttcttcggt 300gttaatgaat ctggtgctga atttggtaac aatgttattc caggtacttt gggtactgat 360tatacttggc catctccatc ttctattgat tttttcgttg gtaagggttt taatactttc 420agagttccat ttttgatgga aagattgtct ccacctgcta ctggtttgac tggtccattt 480gattctactt atttgcaagg tttgaaaact attgtttctt acattactgg taaaggtggt 540tatgctttgg ttgatccaca taactttatg atttacaacg gtgctactat ttctgatact 600aatgcttttc aaacttggtg gcaaaatttg gctgctcaat ttaagactga ttctcatgtt 660gttttcgatg ttatgaatga accacatgat attccagctc aaactgtttt taacttgaac 720caagctgcta ttaatagaat tagagcttct ggtgctactt ctcaatctat tttggttgaa 780ggtacttctt atactggtgc ttggacttgg actactactt ctggtaattc tcaagttttt 840ggtgctattc atgatccaaa caacaatgtt gctattgaaa tgcatcaata cttggattct 900gatggttctg gtacttctcc aacttgtgtt tctccaacta ttggtgctga aagattgcaa 960gctgctactc aatggttgca acaaaacaat ttgaaaggtt tcttgggtga aattggtgct 1020ggttctaatg ctgattgtat ttctgctgtt caaggtgctt tgtgtgaaat gcaacaatct 1080gatgtttggt tgggtgcttt gtggtgggct gctggtccat ggtggggtga ttattttcaa 1140tctattgaac caccatctgg tgttgctgtt tcttctattt tgccacaagc tttggaacca 1200tttttgttat aaggcgcgcc 122052521PRTAspergillus kawachii 52Met Arg Ile Ser Asn Leu Ile Val Ala Ala Ser Ala Ala Thr Met Val 1 5 10 15 Ser Ala Leu Pro Ser Arg Gln Met Lys Lys Arg Asp Ser Gly Phe Lys 20 25 30 Trp Val Gly Thr Ser Glu Ser Gly Ala Glu Phe Gly Ser Ala Leu Pro 35 40 45 Gly Thr Leu Gly Thr Asp Tyr Thr Trp Pro Glu Thr Ser Lys Ile Gln 50 55 60 Val Leu Arg Asn Lys Gly Met Asn Ile Phe Arg Ile Pro Phe Leu Met 65 70 75 80 Glu Arg Leu Thr Pro Asp Gly Leu Thr Gly Ser Phe Ala Ser Thr Tyr 85 90 95 Leu Ser Asp Leu Lys Ser Thr Val Glu Phe Val Thr Asn Ser Gly Ala 100 105 110 Tyr Ala Val Leu Asp Pro His Asn Tyr Gly Arg Phe Asp Gly Ser Ile 115 120 125 Ile Glu Ser Thr Ser Asp Phe Lys Thr Trp Trp Lys Asn Val Ala Thr 130 135 140 Glu Phe Ala Asp Asn Asp Lys Val Ile Phe Asp Thr Asn Asn Glu Tyr 145 150 155 160 His Asp Met Glu Gln Ser Leu Val Leu Asn Leu Asn Gln Ala Ala Ile 165 170 175 Asn Gly Ile Arg Ala Ala Gly Ala Thr Thr Gln Tyr Ile Phe Val Glu 180 185 190 Gly Asn Ala Tyr Thr Gly Ala Trp Asp Trp Thr Thr Tyr Asn Asp Asp 195 200 205 Leu Ser Gly Leu Thr Asp Ser Glu Asp Lys Ile Ile Tyr Glu Met His 210 215 220 Gln Tyr Leu Asp Ser Asp Ser Ser Gly Thr Ser Glu Thr Cys Val Ser 225 230 235 240 Ser Thr Ile Gly Lys Glu Arg Ile Glu Lys Ala Thr Glu Trp Leu Lys 245 250 255 Thr Asn Asn Lys Gln Gly Ile Ile Gly Glu Phe Ala Gly Gly Val Asn 260 265 270 Ser Val Cys Glu Glu Ala Val Glu Gly Met Leu Ala Tyr Met Ser Glu 275 280 285 Asn Ser Asp Val Trp Val Gly Ala Ser Trp Trp Ser Ala Gly Pro Trp 290 295 300 Trp Gly Thr Tyr Met Tyr Ser Leu Glu Pro Thr Asp Gly Thr Ala Tyr 305 310 315 320 Ser Thr Tyr Leu Pro Ile Leu Glu Lys Tyr Phe Pro Ser Gly Asp Ala 325 330 335 Ser Ser Ser Ser Ser Ala Ser Ala Ser Val Ala Ala Ala Thr Ser Ala 340 345 350 Val Ser Thr Thr Thr Thr Ala Ala Phe Glu Gln Thr Thr Thr Pro Ala 355 360 365 Thr Gln Val Glu Ile Ala Ser Ser Ser Ser Ser Ser Ser Ala Val Ala 370 375 380 Ala Ser Gln Thr Thr Leu Ser Lys Val Lys Ser Lys Ser Lys Ser Pro 385 390 395 400 Cys Lys Leu Ser Ser Ala Thr Ser Ser Ala Val Ser Ser Ala Ala Ala 405 410 415 Val Thr Thr Pro Ala Val Ala Ala Thr Thr Pro Ala Ala Ala Pro Thr 420 425 430 Ser Ser Ser Val Ala Phe Ala Thr Thr Ser Val Tyr Val Pro Thr Thr 435 440 445 Thr Ala Ala Ala Pro Ser Gln Val Ser Ser Ser Ala Ala Ala Ser Ser 450 455 460 Ser Gly Val Val Gly Val Ser Asp Pro Gln Gly Pro Ser Ala Thr Asn 465 470 475 480 Ser Ala Gly Glu Val Asn Gln Tyr Tyr Gln Cys Gly Gly Ile Asn Trp 485 490 495 Thr Gly Pro Thr Val Cys Ala Ser Pro Tyr Thr Cys Lys Val Gln Asn 500 505 510 Asp Tyr Tyr Tyr Gln Cys Val Ala Glu 515 520 53419PRTHeterodera schachtii 53Met His Trp Ala Asp Val Ala Cys Ser Arg Pro Pro Trp Pro Arg Asp 1 5 10 15 Ser Val Lys Ala Leu Lys Cys Asn Trp Asn Ala Asn Val Ile Arg Gly 20 25 30 Ala Met Gly Val Asp Glu Gly Gly Tyr Leu Ser Asp Ala Asn Thr Ala 35 40 45 Tyr Asn Leu Met Val Ala Val Ile Glu Ala Ala Ile Ser Asn Gly Ile 50 55 60 Tyr Val Ile Val Asp Trp His Ala His Asn Ala His Pro Asp Glu Ala 65 70 75 80 Val Lys Phe Phe Thr Arg Ile Ala Gln Ala Tyr Gly Ser Tyr Leu His 85 90 95 Ile Leu Tyr Glu Asp Phe Asn Glu Pro Leu Asp Val Ser Trp Thr Asp 100 105 110 Val Leu Val Pro Tyr His Lys Lys Val Ile Ala Ala Ile Arg Ala Ile 115 120 125 Asp Lys Lys Asn Val Ile Ile Leu Gly Thr Pro Lys Trp Ser Gln Asp 130 135 140 Val Asp Val Ala Ser Gln Asn Pro Ile Lys Asp Tyr Gln Asn Leu Met 145 150 155 160 Tyr Thr Leu His Phe Tyr Ala Ser Ser His Phe Thr Ser Asp Leu Gly 165 170 175 Ala Lys Leu Lys Thr Ala Val Asn Asn Gly Leu Pro Val Phe Val Thr 180 185 190 Glu Tyr Gly Thr Cys Glu Ala Ser Gly Asn Gly Asn Leu Asn Thr Asp 195 200 205 Ser Met Ser Ser Trp Trp Thr Leu Leu Asp Ser Leu Lys Ile Ser Tyr 210 215 220 Ala Asn Trp Ala Ile Ser Asp Lys Ser Glu Ala Cys Ser Ala Leu Ser 225 230 235 240 Pro Gly Thr Thr Ala Val Asn Val Gly Val Ser Ser Arg Trp Thr Ser 245 250 255 Ser Gly Asn Met Val Ala Ser Tyr Tyr Lys Lys Lys Ser Thr Gly Ile 260 265 270 Ser Cys Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly 275 280 285 Ser Ser Gly Thr Ser Ser Gly Ser Ser Gly Ser Ser Ser Gly Ser Ser 290 295 300 Ser Gly Ser Ser Ser Gly Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser 305 310 315 320 Gly Ser Gly Ser Ala Ser Ile Ser Val Val Pro Ser Asn Thr Trp Asn 325 330 335 Gly Gly Gly Arg Val Asn Phe Glu Ile Lys Asn Thr Gly Ser Val Pro 340 345 350 Leu Cys Gly Val Val Phe Ser Val Ser Leu Pro Ser Gly Thr Thr Leu 355 360 365 Gly Gly Ser Trp Asn Met Glu Ser Ala Gly Ser Gly Gln Tyr Ser Leu 370 375 380 Pro Ser Trp Val Arg Ile Glu Ala Gly Lys Ser Ser Lys Asp Ala Gly 385 390 395 400 Leu Thr Phe Asn Gly Lys Asp Lys Pro Thr Ala Lys Ile Val Thr Thr 405 410 415 Lys Lys Cys 54418PRTHypocrea jecorina 54Met Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr 1 5 10 15 Gly Gly Ala Val Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile 20 25 30 Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr 35 40 45 Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr 50 55 60 Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr 65 70 75 80 Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala 85 90 95 Gly Val Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100 105 110 Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser 115 120 125 Asn Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130 135 140 Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val 145 150 155 160 Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr 165 170 175 Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val 180 185 190 Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly 195 200 205 Gly Pro Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser 210 215 220 Lys Tyr Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro 225 230 235 240 His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val 245 250 255 Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260 265 270 Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala 275 280 285 Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu 290 295 300 Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His 305 310 315 320 Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala 325 330 335 Thr Trp Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly 340 345 350 Gly Gly Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360 365 Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly 370 375 380 Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Ser 385 390 395 400 Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala 405 410 415 Arg Lys 55471PRTOrpinomyces sp. 55Met Lys Phe Leu Asn Ser Leu Ser Leu Leu Gly Leu Val Ile Ala Gly 1 5 10 15 Cys Glu Ala Met Arg Asn Ile Ser Ser Lys Glu Leu Val Lys Glu Leu 20 25 30 Thr Ile Gly Trp Ser Leu Gly Asn Thr Leu Asp Ala Ser Cys Val Glu 35 40 45 Thr Leu Asn Tyr Ser Lys Asp Gln Thr Ala Ser Glu Thr Cys Trp Gly 50 55 60 Asn Val Lys Thr Thr Gln Glu Leu Tyr Tyr Lys Leu Ser Asp Leu Gly 65 70 75 80 Phe Asn Thr Phe Arg Ile Pro Thr Thr Trp Ser Gly His Phe Gly Asp 85 90 95 Ala Pro Asp Tyr Lys Ile Ser Asp Val Trp Met Lys Arg Val His Glu 100 105 110 Val Val Asp Tyr Ala Leu Asn Thr Gly Gly Tyr Ala Ile Leu Asn Ile 115 120 125 His His Glu Thr Trp Asn Tyr Ala Phe Gln Lys Asn Leu Glu Ser Ala 130 135 140 Lys Lys Ile Leu Val Ala Ile Trp Lys Gln Ile Ala Ala Glu Phe Gly 145 150 155 160 Asp Tyr Asp Glu His Leu Ile Phe Glu Gly Met Asn Glu Pro Arg Lys 165 170 175 Val Gly Asp Pro Ala Glu Trp Thr Gly Gly Asp Gln Glu Gly Trp Asn 180 185 190 Phe Val Asn Glu Met Asn Ala Leu Phe Val Lys Thr Ile Arg Ala Thr 195 200 205 Gly Gly Asn Asn Ala Asn Arg His Leu Met Ile Pro Thr Tyr Ala Ala 210 215 220 Ser Val Asn Asp Gly Ser

Ile Asn Asn Phe Lys Tyr Pro Asn Gly Asp 225 230 235 240 Asp Lys Val Ile Val Ser Leu His Ser Tyr Ser Pro Tyr Asn Phe Ala 245 250 255 Leu Asn Asn Gly Pro Gly Ala Ile Ser Asn Phe Tyr Asp Gly Asn Glu 260 265 270 Ile Asp Trp Val Met Asn Thr Ile Asn Ser Ser Phe Ile Ser Lys Gly 275 280 285 Ile Pro Val Ile Ile Gly Glu Phe Val Ala Met Asn Arg Asp Asn Glu 290 295 300 Asp Asp Arg Glu Arg Trp Gln Glu Tyr Tyr Ile Lys Lys Ala Thr Ala 305 310 315 320 Leu Gly Ile Pro Cys Val Ile Trp Asp Asn Gly Tyr Phe Glu Gly Glu 325 330 335 Gly Glu Arg Phe Gly Ile Ile Asp Arg Lys Ser Leu Asn Val Ile Phe 340 345 350 Pro Lys Leu Ile Asn Gly Leu Met Lys Gly Leu Gly Asp Glu Lys Pro 355 360 365 Lys Thr Thr Ile Arg Arg Thr Thr Thr Thr Thr Val Gln Val Gln Pro 370 375 380 Thr Ile Asn Asn Glu Cys Phe Ser Thr Arg Leu Gly Tyr Ser Cys Cys 385 390 395 400 Asn Gly Phe Asp Val Leu Tyr Thr Asp Asn Asp Gly Gln Trp Gly Val 405 410 415 Glu Asn Gly Asn Trp Cys Gly Ile Lys Ser Ser Cys Gly Asn Asn Gln 420 425 430 Arg Gln Cys Trp Ser Glu Arg Leu Gly Tyr Pro Cys Cys Gln Tyr Thr 435 440 445 Thr Asn Ala Glu Tyr Thr Asp Asn Asp Gly Arg Trp Gly Val Glu Asn 450 455 460 Gly Asn Trp Cys Gly Ile Tyr 465 470 56399PRTIrpex lacteus 56Met Lys Ser Leu Leu Leu Ser Ala Ala Ala Thr Leu Ala Leu Ser Thr 1 5 10 15 Pro Ala Phe Ser Val Ser Val Trp Gly Gln Cys Gly Gly Ile Gly Phe 20 25 30 Thr Gly Ser Thr Thr Cys Asp Ala Gly Thr Ser Cys Val His Leu Asn 35 40 45 Asp Tyr Tyr Phe Gln Cys Gln Pro Gly Ala Ala Thr Ser Thr Val Gln 50 55 60 Pro Thr Thr Thr Ala Ser Ser Thr Ser Ser Ala Ala Ala Pro Ser Ser 65 70 75 80 Ser Gly Asn Ala Val Cys Ser Gly Thr Arg Asn Lys Phe Lys Phe Phe 85 90 95 Gly Val Asn Glu Ser Gly Ala Glu Phe Gly Asn Asn Val Ile Pro Gly 100 105 110 Thr Leu Gly Thr Asp Tyr Thr Trp Pro Ser Pro Ser Ser Ile Asp Phe 115 120 125 Phe Val Gly Lys Gly Phe Asn Thr Phe Arg Val Pro Phe Leu Met Glu 130 135 140 Arg Leu Ser Pro Pro Ala Thr Gly Leu Thr Gly Pro Phe Asp Ser Thr 145 150 155 160 Tyr Leu Gln Gly Leu Lys Thr Ile Val Ser Tyr Ile Thr Gly Lys Gly 165 170 175 Gly Tyr Ala Leu Val Asp Pro His Asn Phe Met Ile Tyr Asn Gly Ala 180 185 190 Thr Ile Ser Asp Thr Asn Ala Phe Gln Thr Trp Trp Gln Asn Leu Ala 195 200 205 Ala Gln Phe Lys Thr Asp Ser His Val Val Phe Asp Val Met Asn Glu 210 215 220 Pro His Asp Ile Pro Ala Gln Thr Val Phe Asn Leu Asn Gln Ala Ala 225 230 235 240 Ile Asn Arg Ile Arg Ala Ser Gly Ala Thr Ser Gln Ser Ile Leu Val 245 250 255 Glu Gly Thr Ser Tyr Thr Gly Ala Trp Thr Trp Thr Thr Thr Ser Gly 260 265 270 Asn Ser Gln Val Phe Gly Ala Ile His Asp Pro Asn Asn Asn Val Ala 275 280 285 Ile Glu Met His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Pro 290 295 300 Thr Cys Val Ser Pro Thr Ile Gly Ala Glu Arg Leu Gln Ala Ala Thr 305 310 315 320 Gln Trp Leu Gln Gln Asn Asn Leu Lys Gly Phe Leu Gly Glu Ile Gly 325 330 335 Ala Gly Ser Asn Ala Asp Cys Ile Ser Ala Val Gln Gly Ala Leu Cys 340 345 350 Glu Met Gln Gln Ser Asp Val Trp Leu Gly Ala Leu Trp Trp Ala Ala 355 360 365 Gly Pro Trp Trp Gly Asp Tyr Phe Gln Ser Ile Glu Pro Pro Ser Gly 370 375 380 Val Ala Val Ser Ser Ile Leu Pro Gln Ala Leu Glu Pro Phe Leu 385 390 395 5742PRTArtificial SequenceDescription of Artificial Sequence Synthetic flexible linker polypeptide 57Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Trp His Pro Gln Phe 1 5 10 15 Gly Gly Glu Asn Leu Tyr Phe Gln Gly Asp Tyr Lys Asp Asp Asp Lys 20 25 30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 5837PRTArtificial SequenceDescription of Artificial Sequence Synthetic flexible linker polypeptide 58Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Trp Ser His Pro Gln Phe 1 5 10 15 Glu Lys Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Gly Gly Gly Ser 20 25 30 Gly Gly Gly Gly Ser 35 5931DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59gactgaattc ataatggtct ccttcacctc c 316034DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60gactctcgag ttacaaacat tgagagtagt atgg 346131DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61cagtctcgag ttacaagaaa gatgggttag c 316263DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 62gcgttggtac cgtttaaacg gggcccttaa ttaaacaatg ctaagaagag ctttactatt 60gag 636340DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 63cctcccccgg gttagaagca gtgaaagtgg agttgattgg 406442DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 64gcgacgagtc aaccctccag gtggtaacag aggtactacc ac 426546DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 65gcgactcgag ggcgcgccta caaacattga gagtagtatg ggttta 466647DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 66gcgttgagct cgggccctaa tttttatttt agattcctga cttcaac 476745DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 67gcgttgaatt cttaattaag taaaaagtag ataattactt ccttg 456847DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 68gcgttgaatt cttaattaaa caatgattgt cggcattctc accacgc 476945DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 69gcgatgaatt cggcgcgcct tacaggaacg atgggtttgc gtttg 457031DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70gatcggatcc caattaatgt gagttacctc a 317134DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71gtacaagctt agatctccta tgcggtgtga aata 347221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 72atttcttctt gaaccatgaa c 217322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 73cttaatcaag acttctgtag cc 22

Claims

1-211. (canceled)

212. A host cell comprising at least one heterologous polynucleotide comprising a nucleic acid encoding a cellulose, wherein the cellulase has an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 54.

213. The host cell of claim 212, wherein the cellulase has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 54.

214. The host cell of claim 212, wherein the cellulase has an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 54.

215. The host cell of claim 212, wherein the nucleic acid is at least 90% identical to the nucleotide sequence of SEQ ID NO: 49.

216. The host cell of claim 212, wherein the nucleic acid is codon-optimized.

217. The host cell of claim 212, wherein the host cell is a Saccharomyces cerevisiae host cell.

218. The host cell of claim 212, wherein the host cell is a Kluyveromyces host cell.

219. The host cell of claim 212, wherein the host cell is thermotolerant.

220. The host cell of claim 212, wherein the host cell is a xylose-utilizing host cell.

221. The host cell of claim 212, wherein the host cell can hydrolyze Avicel.

222. The host cell of claim 212, wherein the host cell further comprises a heterologous polynucleotide comprising a nucleic acid encoding a .beta.-glucosidase I, a heterologous polynucleotide comprising a nucleic acid encoding a cellobiohydrolase I; and a heterologous polynucleotide comprising a nucleic acid encoding a cellobiohydrase II.

223. The host cell of claim 222, wherein the host cell further comprises a heterologous polynucleotide comprising a nucleic acid encoding an S. fibuligera .beta.-glucosidase I, a heterologous polynucleotide comprising a nucleic acid encoding a T. emersonii cellobiohydrolase I; and a heterologous polynucleotide comprising a nucleic acid encoding a C. lucknowense cellobiohydrase II.

224. The host cell of claim 222, wherein the host cell can produce ethanol when grown using cellulose as a carbon source.

225. A method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell according to claim 212.

226. The method of claim 225, wherein said cellulosic substrate is a lignocellulosic biomass selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, Agave, and combinations thereof.

227. The method of claim 225, further comprising contacting said cellulosic substrate with externally produced cellulase enzymes.

228. The method of claim 225, wherein said host cell produces ethanol.

229. The method of claim 228, wherein ethanol is produced at a rate of at least about 10 mg per hour per liter.

230. A method of fermenting cellulose using the host cell of claim 212, said method comprising culturing said transformed host cell in medium that contains insoluble cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose.

231. The method of claim 230, wherein said culturing is at a temperature of at least about 37.degree. C.