WO2008057637A2

WO2008057637A2 - Methods of increasing secretion of polypeptides having biological activity

Info

Publication number: WO2008057637A2
Application number: PCT/US2007/074038
Authority: WO
Inventors: Sandra Merino
Original assignee: Novozymes, Inc.
Priority date: 2006-07-21
Filing date: 2007-07-20
Publication date: 2008-05-15
Also published as: US20140011256A1; EP2046819A2; US8871486B2; US20150044756A1; CA2658610A1; US20090298126A1; US8735549B2; DK2046819T3; WO2008057637A3; US9006409B2; EP2046819B1; CN101516906B; ES2538360T3; BRPI0714870A2; US8546106B2; US20140248671A1; WO2008057637A9; CN101516906A

Abstract

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Description

METHODS OF INCREASING SECRETION OF POLYPEPTIDES HAVING BIOLOGICAL ACTIVITY

Statement as to Rights to Inventions Made Under

Federally Sponsored Research and Development

This invention was made with Government support under NREL Subcontract No ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by the Department of Energy The government has certain rights in this invention

Background of the Invention

Field of the Invention

The present invention relates to methods of producing secreted polypeptides having biological activity and to fusion proteins and polynucleotides thereof

Description of the Related Art

The recombinant production of a heterologous polypeptide in a fungal host cell, particularly a filamentous fungal cell such as Aspergillus or Trichoderma or a yeast cell such Saccharomyces, may provide for a more desirable vehicle for producing the polypeptide in commercially relevant quantities

Recombinant production of a secreted heterologous polypeptide is generally accomplished by constructing an expression cassette in which the DNA coding for the polypeptide is operably linked to a promoter suitable for the host cell and a signal peptide coding region that codes for an amino acid sequence linked in frame to the amino terminus of the polypeptide and directs the encoded polypeptide into the cell's secretory pathway The expression cassette is introduced into the host cell, usually by plasmid-mediated transformation Production of the secreted heterologous protein is then achieved by cultunng the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette

While expression of a heterologous polypeptide in a host cell may be improved, an obstacle often encountered is that the polypeptide is poorly secreted into the culture medium One method of improving secretion of the polypeptide is to replace the native signal peptide coding sequence with a foreign signal peptide coding region to enhance secretion of the polypeptide However, in some cases, such a replacement does not provide a sufficient improvement for producing the polypeptide in commercially relevant quantities Another method is to fuse the polypeptide to another polypeptide that is highly secreted by a host cell The highly secreted polypeptide functions as a earner to transport the poorly secreted or non-secreted polypeptide as a fusion protein through the cell's secretory pathway

5 WO 05/093050 discloses a fusion protein composed of an exo-cellobiohydrolase catalytic domain and a cellulase catalytic domain to increase the yield of a cellulase enzyme Gouka et al , 1997, Applied and Environmental Microbiology Feb 1997, p 488-497, discloses glucoamylase gene fusions that alleviate limitations for protein production in Aspergillus awamon Nyyssonen and Keranen, 1995 Current Genetics 10 28 71-79, discloses multiple roles of the cellobiohydrolase I in enhancing production of fusion antibodies by Tnchoderma reesei

It is an object of the present invention to provide methods for increasing the secretion of polypeptides having biological activity

I₅ Summary of the Invention

The present invention relates to methods for producing a secreted polypeptide having biological activity, compπsing

(a) transforming a fungal host cell with a fusion protein construct encoding a 20 fusion protein, wherein the fusion protein construct comprises

(ι) a first polynucleotide compπsing a nucleotide sequence encoding a signal peptide,

(ιι) a second polynucleotide compπsing a nucleotide sequence encoding at least a catalytic domain of an endoglucanase or a portion thereof, 2₅ and

(iii) a third polynucleotide comprising a nucleotide sequence encoding at least a catalytic domain of a polypeptide having biological activity or a portion thereof, wherein the signal peptide and at least the catalytic domain of the 30 endoglucanase or the portion thereof increases secretion of the polypeptide having biological activity or the portion thereof compared to the absence of at least the catalytic domain of the endoglucanase or the portion thereof,

(b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein, and

3₅ (C) recoveπng the fusion protein, a component thereof, or a combination thereof, from the cultivation medium, wherein the fusion protein or the component thereof has biological activity In one aspect, the 3' end of the first polynucleotide is operably linked to the 5' end of the second polynucleotide and the 3' end of the second polynucleotide is operably linked to the 5' end of the third polynucleotide or the 3' end of the first polynucleotide is operably linked to the 5' end of the third polynucleotide and the 3' end of the third polynucleotide is operably linked to the 5' end of the second polynucleotide to encode a fusion protein

The present invention also relates to isolated fusion proteins, compnsing

(a) a first amino acid sequence compnsing a signal peptide,

(b) a second amino acid sequence compnsing at least a catalytic domain of an endoglucanase or a portion thereof, and

(c) a third amino acid sequence comprising at least a catalytic domain of a polypeptide having biological activity or a portion thereof

The present invention also relates to polynucleotides encoding the fusion proteins, and fusion protein constructs, expression vectors, and recombinant host cells compnsing such polynucleotides

In another aspect, the C-terminal end of the first amino acid sequence is linked in frame to the N-terminal end of the second amino acid sequence and the C-terminal end of the second ammo acid sequence is linked in frame to the N-terminal end of the third amino acid sequence or the C-terminal end of the first amino acid sequence is linked in frame to the N-terminal end of the third amino acid sequence and the C-terminal end of the third amino acid sequence is linked in frame to the N-terminal end of the second amino acid sequence

The present invention further relates to methods of using the fusion proteins or components thereof

Brief Description of the Figures

Figure 1 shows a restriction map of pMJ04

Figure 2 shows a restriction map of pCaHj527 Figure 3 shows a restriction map of pMT2188

Figure 4 shows a restriction map of pCaHj568

Figure 5 shows a restriction map of pMJ05

Figure 6 shows a restriction map of pSMaι130

Figure 7 shows the DNA sequence and amino acid sequence of an Aspergillus or/zae beta-glucosidase native signal sequence (SEQ ID NOs 59 and 60)

Figure 8 shows the DNA sequence and amino acid sequence of a Humicola insolens endoglucanase V signal sequence (SEQ ID NOs 63 and 64) Figure 9 shows a restriction map of pSMaι135

Figure 10 shows a restriction map of pSMaι140

Figure 11 shows a restriction map of pSaMe-Fi

Figure 12 shows a restriction map of pSaMe-FX Figure 13 shows a restriction map of pAILo47

Figures 14A, 14B, 14C, and 14D show the DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase variant fusion protein (SEQ ID NOs 73 and 74, respectively)

Figures 15A, 15B, 15C, and 15D show the DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase fusion protein (SEQ ID NOs 75 and 76, respectively)

Definitions

Endoglucanase activity: The term "endoglucanase activity" is defined herein as an endo-1,4-beta-D-glucan 4-glucanohydrolase (E C 32 1 4), which catalyses endohydrolysis of 1,4-beta-D-glycosιdιc linkages in cellulose, cellulose denvatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta- 1,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure and Appl Chem 59 257-268 One unit of endoglucanase activity is defined as 1 0 μmole of reducing sugars produced per minute at 50°C, pH 48

Beta-glucosidase activity: The term "beta-glucosidase" is defined herein as a beta-D-glucoside glucohydrolase (E C 32 1 21), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose For purposes of the present invention, beta-glucosidase activity is determined according to the procedure described by Ventuπ et al , 2002, J Basic Microbiol 42 55-66, except different conditions are employed as descnbed herein One unit of beta-glucosidase activity is defined as 1 0 μmole of p-nitrophenol produced per minute at 50⁰C, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate 0 01% TWEEN® 20

Full-length polypeptide: The term "full-length polypeptide" is defined herein as a precursor form of a polypeptide having biological activity, wherein the precursor contains a signal peptide region and alternatively also a propeptide region, wherein upon secretion from a cell, the signal peptide is cleaved and alternatively also the propeptide is cleaved yielding a polypeptide with biological activity Signal peptide: The term "signal peptide" is defined herein as a peptide linked in frame to the amino terminus of a polypeptide and directs the encoded polypeptide into a cell's secretory pathway

Signal peptide coding sequence: The term "signal peptide coding sequence" is defined herein as a peptide coding region that codes for an amino acid sequence linked in frame to the amino terminus of an encoded polypeptide and directs the encoded polypeptide into a cell's secretory pathway

Propeptide: The term "propeptide" is defined herein as a peptide linked in frame to the amino terminus of a polypeptide The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases) A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is linked in frame to the amino terminus of a polypeptide and the signal peptide region is linked in frame to the amino terminus of the propeptide region

Propeptide coding sequence: The term "propeptide coding sequence" is defined herein as a peptide coding region that codes for an amino acid sequence linked in frame to the amino terminus of a polypeptide forming a proenzyme or propolypeptide (or a zymogen in some cases) Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide having biological activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc

Catalytic domain: The term "catalytic domain" is defined herein as a structural portion or region of the amino acid sequence of an endoglucanase or a polypeptide having biological activity (e g , beta-glucosidase activity), which possesses the catalytic activity of the endoglucanase or the polypeptide having biological activity (e g , beta- glucosidase) The catalytic domain is also referred to as the "core" region herein

Fusion protein: The term "fusion protein" is defined herein as a polypeptide that exhibits biological activity and that comprises at least both an endoglucanase catalytic domain and a catalytic domain of a polypeptide having biological activity (e g , beta- glucosidase)

Beta-glucosidase fusion protein: The term "beta-glucosidase fusion protein" is defined herein as a polypeptide that exhibits beta-glucosidase activity and that compπses at least both a beta-glucosidase catalytic domain and an endoglucanase catalytic domain Components of a fusion protein: The term "components of a fusion protein" is defined herein as individual (cleaved) fragments of the fusion protein, wherein each fragment has biological activity and includes either at least the catalytic domain of a endoglucanase and at least the catalytic domain of a polypeptide having biological activity or at least the catalytic domain of a polypeptide having biological activity For example, the presence of a cleavage site, e g , Kex2 site, between the components of at least the catalytic domain of a endoglucanase and at least the catalytic domain of a polypeptide having biological activity of the fusion protein can result in the production of a polypeptide having endoglucanase activity and another polypeptide having biological activity

Components of a beta-glucosidase fusion protein: The term "components of a beta-glucosidase fusion protein" is defined herein as individual (cleaved) fragments of the beta-glucosidase fusion protein, wherein each fragment has beta-glucosidase activity and is either at least the catalytic domain of the endoglucanase and at least the beta-glucosidase catalytic domain or at least the beta-glucosidase catalytic domain For example, the presence of a cleavage site, e g , Kex2 site, between the endoglucanase and beta-glucosidase components of the fusion protein can result in the production of a polypeptide having endoglucanase activity and another polypeptide having beta- glucosidase activity Carbohydrate binding module: The term "carbohydrate binding module

(CBM)" is defined herein as a portion of the amino acid sequence of an endoglucanase (cellulase) that is involved in the binding of the endoglucanase to cellulose (lignocellulose) Carbohydrate binding modules generally function by non-covalently binding the endoglucanase to cellulose, a cellulose deπvative, or a polysaccharide equivalent thereof CBMs typically function independent of the catalytic domain

Fusion protein construct: The term "fusion protein construct" refers to a nucleic acid construct that is composed of different genes or portions thereof in operable linkage The components include from the 5' end a DNA molecule encoding at least an endoglucanase catalytic domain and a DNA molecule encoding at least a catalytic domain of a polypeptide having biological activity

Beta-glucosidase fusion construct: The term "beta-glucosidase fusion construct" refers to a nucleic acid construct that is composed of different genes or portions thereof in operable linkage The components include from the 5' end a DNA molecule encoding at least an endoglucanase catalytic domain and a DNA molecule encoding at least a beta-glucosidase catalytic domain

Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE

Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0 5% by weight of other polypeptide matenal with which it is natively or recombinants associated It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99 5% pure, and even most preferably 100% pure by weight of the total polypeptide matenal present in the preparation The polypeptides of the present invention are preferably in a substantially pure form, / e , that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinant^ associated This can be accomplished, for example, by prepanng the polypeptide by means of well-known recombinant methods or by classical purification methods

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is descnbed by the parameter "identity^*

For purposes of the present invention, the degree of identity between two ammo acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J MoI Biol 48 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS The European Molecular Biology Open Software Suite, Rice et al , 2000, Trends in Genetics 16 276-277), preferably version 3 00 or later The optional parameters used are gap open penalty of 10, gap extension penalty of 0 5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matπx The output of Needle labeled "longest identity" (obtained using the -nobnef option) is used as the percent identity and is calculated as follows (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm

(Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the

EMBOSS package (EMBOSS The European Molecular Biology Open Software Suite, Rice et al , 2000, supra), preferably version 3 0 0 or later The optional parameters used are gap open penalty of 10, gap extension penalty of 05, and the EDNAFULL (EMBOSS version of NCBI NUC44) substitution matrix The output of Needle labeled "longest identity" (obtained using the -πobπef option) is used as the percent identity and is calculated as follows

(Identical Deoxyπbonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment) Polypeptide fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of a fusion protein (e g , beta-glucosidase fusion protein) or components thereof, wherein the fragment has biological activity (e g , beta-glucosidase activity)

Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5' and/or 3' end of a polynucleotide, wherein the subsequence encodes a polypeptide fragment having biological activity, e g , beta-glucosidase activity or endoglucanase activity

Isolated polynucleotide: The term "isolated polynucleotide" as used herein refers to a polynucleotide that is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by agarose electrophoresis

Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0 5% by weight of other polynucleotide material with which it is natively or recombinant^ associated A substantially pure polynucleotide may, however, include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 995% pure by weight The polynucleotides are preferably in a substantially pure form, i e , that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinant^ associated The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof cDNA: The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell cDNA lacks iπtroπ sequences that are usually present in the corresponding genomic DNA The initial, primary RNA transcript is a precursor to mRNA that is processed through a seπes of steps before appearing as mature spliced mRNA These steps include the removal of intron sequences by a process called splicing cDNA deπved from mRNA lacks, therefore, any intron sequences

Nucleic acid construct: The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence

Control sequence: The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator At a minimum, the control sequences include a promoter, and transcπptional and translational stop signals The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide

Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropπate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide In addition, the term "operably linked" also relates to two polynucleotides that are linked or fused, which are expressed together as a fused or fusion protein

Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the ammo acid sequence of its protein product The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA The coding sequence may be a DNA, cDNA, or recombinant nucleotide sequence

Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcnption, post-transcπptional modification, translation, post-translational modification, and secretion

Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention and is operably linked to additional nucleotides that provide for its expression

Host cell: The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide

Variant: When used herein, the term "variant^* means a polypeptide having biological activity produced by an organism expressing a modified nucleotide sequence, e g , SEQ ID NO 25 or a homologous sequence thereof, or the mature coding region thereof The modified nucleotide sequence is obtained through human intervention by modification of a nucleotide sequence, e g SEQ ID NO 23 or a homologous sequence thereof, or the mature coding region thereof The modification can be a substitution, a deletion, and/or an insertion of one or more amino acids as well as a replacement of one or more ammo acid side chains

Detailed Description of the Invention

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, wherein the fusion protein construct compnses (ι) a first polynucleotide comprising a nucleotide sequence encoding a signal peptide, (ιι) a second polynucleotide comprising a nucleotide sequence encoding at least a catalytic domain of an endoglucanase or a portion thereof, and (in) a third polynucleotide compnsing a nucleotide sequence encoding at least a catalytic domain of a polypeptide having biological activity or a portion thereof, wherein the signal peptide and at least the catalytic domain of the endoglucanase or the portion thereof increases secretion of the polypeptide having biological activity or the portion thereof compared to the absence of at least the catalytic domain of the endoglucanase or the portion thereof, (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein, and (c) recoveπng the fusion protein, a component thereof, or a combination thereof, from the cultivation medium, wherein the fusion protein or the component thereof has biological activity

In a preferred aspect, the 3' end of the first polynucleotide is operably linked to the 5' end of the second polynucleotide and the 3' end of the second polynucleotide is operably linked to the 5' end of the third polynucleotide In another preferred aspect, the 3' end of the first polynucleotide is operably linked to the 5' end of the third polynucleotide and the 3' end of the third polynucleotide is operably linked to the 5' end of the second polynucleotide to encode a fusion protein

A fusion protein is produced by fusing a nucleotide sequence encoding a polypeptide having biological activity or a portion thereof to a nucleotide sequence encoding a polypeptide having endoglucanase activity or a portion thereof and a nucleotide sequence encoding a signal peptide operably linked to the nucleotide sequence encoding the polypeptide having endoglucanase activity or a portion thereof Techniques for producing fusion proteins are known in the art, and include, for example, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoters) and terminator Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al , 1993, EMBO J 12 2575-2583, Dawson et al , 1994, Science 266 776-779) A fusion protein having biological activity comprising a signal peptide, at least the catalytic domain of an endoglucanase or a portion thereof, and at least the catalytic domain of a polypeptide having biological activity or a portion thereof, increases secretion of the fusion protein compared to the absence of at least the catalytic domain of the endoglucanase or a portion thereof The increase in secretion of the fusion protein having biological activity is at least 5%, preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, more preferably at least 100%, even more preferably at least 150%, even more preferably at least 200%, most preferably at least 500%, and even most preferably at least 1000% compared to the absence of at least the catalytic domain of the endoglucanase

In each of the preferred aspects below, the components of a fusion protein construct (nucleic acid construct) are operably linked from the 5' end to the 3' end of the construct

In a preferred aspect, the fusion protein construct comprises a polynucleotide compnsing a nucleotide sequence encoding a signal peptide, a polynucleotide compπsing a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide compπsing a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide compnsing a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide compnsing a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide compnsing a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide compnsing a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide compnsing a nucleotide sequence encoding a full- length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide compnsing a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide compnsing a nucleotide sequence encoding a full- length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide compnsing a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of an endoglucanase (signal peptide and mature polypeptide), and a polynucleotide compnsing a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity and a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of an endoglucanase (signal peptide and mature polypeptide)

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compnsing a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compnsing a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compπsing a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compπsing a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compπsing a nucleotide sequence encoding a catalytic domain of an endoglucanase In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide comprising a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide compπsing a nucleotide sequence encoding a full-length polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, and a polynucleotide compπsing a nucleotide sequence encoding a mature polypeptide of an endoglucanase In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, a polynucleotide compnsing a nucleotide sequence encoding another signal peptide, and a polynucleotide compnsing a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, a polynucleotide compnsing a nucleotide sequence encoding another signal peptide, and a polynucleotide compnsing a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, a polynucleotide compnsing a nucleotide sequence encoding another signal peptide, and a polynucleotide compπsing a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity; a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, a polynucleotide compnsing a nucleotide sequence encoding another signal peptide, and a polynucleotide compnsing a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein construct comprises a polynucleotide comprising a nucleotide sequence encoding a signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of a polypeptide having biological activity, and a polynucleotide encoding a linker and/or a polynucleotide encoding a carbohydrate binding module, a polynucleotide comprising a nucleotide sequence encoding another signal peptide, and a polynucleotide comprising a nucleotide sequence encoding a mature polypeptide of an endoglucanase

In another preferred aspect, for each of the preferred aspects above, the polynucleotides may encode a portion of the catalytic domain, the mature polypeptide, or the full-length polypeptide of an endoglucanase or a polypeptide having biological activity The portion of the endoglucanase may or may not have endoglucanase activity In a more preferred aspect, the portion of the endoglucanase has endoglucanase activity

In each of the preferred aspects above, the components of the fusion protein constructs further comprise a promoter region and/or a terminator region

Endoglucanases and Polynucleotides Thereof

A polynucleotide encoding a catalytic domain, mature polypeptide, or full-length polypeptide of an endoglucanase, or portions thereof, may be obtained from any organism For purposes of the present invention, the term "polypeptide" will be understood to include a full-length polypeptide, mature polypeptide, or catalytic domain, or portions or fragments thereof that have activity The term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted Many endoglucanases have a multidomain structure consisting of a catalytic domain separated from a carbohydrate binding domain (CBM) by a linker peptide (Suurnakki et al , 2000, Cellulose 7 18Θ-209) The catalytic domain contains the active site whereas the CBM interacts with cellulose by binding the enzyme to it (van Tilbeurgh ef al , 1986, FESS Letters 204 223-227, Tomme et al , 1988, European Journal of Biochemistry i70 575-581)

A polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding a bacterial polypeptide For example, the polypeptide may be a Gram positive bacterial polypeptide including, but not limited to, a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobaαllus, or Oceanobaαllus polypeptide, e g , a Bacillus alkalophilus, Bacillus amyloliquefaciens. Bacillus brews. Bacillus αrculans. Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megatenum, Bacillus stearothermophilus, Bacillus subtώs. Bacillus thuπngiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus ubens, and Streptococcus equi subsp Zooepidemicus, Streptomyces In/idans, or Streptomyces murinus polypeptide, or a Gram negative bacterial polypeptide including, but not limited to, an E coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobactenum, Fusobacteπum, llyobacter, Neisseria, or Ureaplasma polypeptide

Examples of bacteπal endoglucanases that can be used as sources for the polynucleotides in the methods of the present invention, include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039, WO 93/15186, U S Patent No 5,275,944, WO 96/02551 , U S Patent No 5,536,655, WO 00/70031 , WO 05/093050), Thermobifida fusca endoglucanase III (WO 05/093050), and Thermobifida fusca endoglucanase V (WO 05/093050)

A polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Ktuyveromyces, Pichia, Sacchammyces, Schizosaccharomyces, or Yarrowia polypeptide, or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cladorrhinum, Cryptococcus, Filibasidium, Fusanum, Humicola, Magnaporthe, Mucor, Mycehophthora, Neocallimastix, Neurospora, Paeαlomyces, Pemαllium, Pimmyces, Schizophyltum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Tπchodema polypeptide

In a preferred aspect, a polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding a Sacchammyces carlsbergensis, Sacchammyces cerevisiae, Sacchammyces diastaticus, Saccharomyces douglasn, Sacchammyces kluyveπ, Sacchammyces norbensis, or Sacchammyces oviformis polypeptide

In another preferred aspect, a polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding an Aspergillus aculeatus, Aspergillus awamoπ, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Cladorrhinum foecundissimum, Fusanum bactπdioides, Fusanum cerealis, Fusanum crookwellense, Fusanum culmorum, Fusanum graminearum, Fusanum graminum, Fusanum heterosporum, Fusanum negundi, Fusanum oxysporum, Fusanum reticulatum, Fusanum mseum, Fusanum sambuαnum, Fusanum sarcochmum, Fusanum spomtπchioides, Fusanum sulphureum, Fusanum torulosum, Fusanum tnchothecioides, Fusanum venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Mycehophthora thermophila, Neurospora crassa, Pemαllium purpumgenum, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia sub thermophila, Thielavia terrestns, Thielavia temcola, Thielavia thermophila, Thielavia vanospora, Thielavia waremgii, Tπchoderma harzianum, Tnchoderma komngii, Tπchoderma longibrachiatum, Tnchoderma reesei, or Tnchoderma vinde polypeptide Examples of fungal endoglucaπases that can be used as sources for the polynucleotides in the methods of the present invention, include, but are not limited to, a Tnchoderma reesei EG1 (Penttila et al , 1986, Gene 45 253-263, GENBANK'" accession no M15665), Tnchoderma reesei EG2 (Saloheimo, et al , 1988, Gene 63 11- 22, GENBANK™ accession no M19373), Tnchoderma reesei EG3 (Okada et al , 1988, Appl Environ Microbiol 64 555-563, GENBANK™ accession no AB003694), Tnchoderma reesei EG4 (Saloheimo et al , 1997, Eur J Biochem 249 584-591 , GENBANK™ accession no Y11113), and Tnchoderma reesei EG5 (Saloheimo et al , 1994, Molecular Microbiology 13 219-228, GENBANK™ accession no Z33381), Aspergillus aculeatus endoglucanase (Ooi et al , 1990, Nucleic Acids Research 18 5884), Aspergillus kawachn (Sakamoto et al , 1995, Current Genetics 27 435-439), Erwinia carotovara endoglucanase (Saaπlahti ef al , 1990, Gene 90 9-14), Fusarium oxysporum (GENBANK™ accession no L29381), Humicola gπsea var thermoidea (GENBANK™ accession no AB003107), Melanocarpυs albomyces (GENBANK™ accession no MAL515703), and Neurospora crassa (GENBANK™ accession no XM_324477 )

Other endoglucanases are disclosed in numerous Glycosyl Hydrolase families using the classification according to Menπssat B , 1991 , A classification of glycosyl hydrolases based on ammo-acid sequence similarities, Biochem J 280 309-316, and Henrissat B , and Bairoch A , 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem J 316 695-696

The techniques used to isolate or clone a polynucleotide encoding a polypeptide having endoglucanase activity are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof The cloning of the polynucleotides from such genomic DNA can be effected, e g , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features See, e g , lnnis et al , 1990, PCR A Guide to Methods and Application, Academic Press, New York Other nucleic acid amplification procedures such as ligase chain reaction (LCR), hgated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used

It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e g , anamorphs, regardless of the species name by which they are known Those skilled in the art will readily recognize the identity of appropriate equivalents Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL)

In a preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase I gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase Il gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase III gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase IV gene In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase V gene In a more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene that encodes the polypeptide of SEQ ID NO 2 In another most preferred aspect, the full-length polypeptide of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene that encodes the full-length polypeptide of SEQ ID NO 2 In another most preferred aspect, the mature polypeptide of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene that encodes the mature polypeptide of SEQ ID NO 2 In another most preferred aspect, the catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene that encodes the catalytic domain of SEQ ID NO 2 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene compπsing SEQ ID NO 1 In another most preferred aspect, the full-length polypeptide of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene compnsing SEQ ID NO 1 In another most preferred aspect, the mature polypeptide of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene comprising the mature polypeptide coding sequence of SEQ ID NO 1 In another most preferred aspect, the catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene comprising the catalytic domain coding sequence of SEQ ID NO 1

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from an endoglucanase Vl gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 5 endoglucanase gene In a more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Mycehophthora thermophila CBS 117 65 endoglucanase gene In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Mycehophthora thermophila CBS 117 65 endoglucanase gene that encodes the polypeptide of SEQ ID NO 4 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Myceliophthora thermophila CBS 117 65 endoglucanase gene comprising SEQ ID NO 3 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 495 95 endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 495 95 endoglucanase gene that encodes the polypeptide of SEQ ID NO 6 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 495 95 endoglucanase gene comprising SEQ ID NO 5 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 494 95 endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 494 95 eπdoglucanase gene that encodes the polypeptide of SEQ ID NO 8 In another most preferred aspect, the full- length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a basidiomycete CBS 494 95 endoglucanase gene comprising SEQ ID NO 7

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 6 endoglucanase gene In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6B endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestπs NRRL 8126 CEL6B endoglucanase gene that encodes the polypeptide of SEQ ID NO 10 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestπs NRRL 8126 CEL6B endoglucanase gene comprising SEQ ID NO 9 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6C endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6C endoglucanase gene that encodes the polypeptide of SEQ ID NO 12 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6C endoglucanase gene comprising SEQ ID NO 11

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 7 endoglucanase gene In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene that encodes the polypeptide of SEQ ID NO 14 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestπs NRRL 8126 CEL7C endoglucanase gene comprising SEQ ID NO 13 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestns NRRL 8126 CEL7E endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestns NRRL 8126 CEL7E endoglucanase gene that encodes the polypeptide of SEQ ID NO 16 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestns NRRL 8126 CEL7E endoglucanase gene comprising SEQ ID NO 15 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia tenrestris NRRL 8126 CEL7F endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia tenrestns NRRL 8126 CEL7F endoglucanase gene that encodes the polypeptide of SEQ ID NO 18 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Thielavia terrestns NRRL 8126 CEL7F endoglucanase gene comprising SEQ ID NO 17 In another more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase gene that encodes the polypeptide of SEQ ID NO 20 In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase gene compnsing SEQ ID NO 19

In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Tnchoderma reesei strain No VTT-D-80133 endoglucanase gene that encodes the polypeptide of SEQ ID NO 22 (GENBANK™ accession no M15665) In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Tnchoderma reesei strain No VTT-D- 80133 endoglucanase gene comprising SEQ ID NO 21 (GENBANK™ accession no M 15665)

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 9 endoglucanase gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 12 endoglucanase gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 45 endoglucanase gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 74 endoglucanase gene In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide obtained from a gene encoding a homologous polypeptide comprising an amino acid sequence that has a degree of identity to the amino acid sequences of the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, or SEQ ID NO 22 of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably 96%, 97%, 98% or 99%, which have endoglucanase activity In a preferred aspect, the homologous polypeptide has an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, or SEQ ID NO 22 In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase, or a portion thereof, is encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under very low stπngency conditions, preferably low stringency conditions, more preferably medium stπngency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stnngency conditions with (ι) SEQ ID NO 1 , SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19 or SEQ ID NO 21, (ιι) the cDNA sequence contained in SEQ ID NO 1 , SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, or SEQ ID NO 21 , (in) a subsequence of (ι) or (ιι), or (ιv) a complementary strand of (ι), (ιι), or (in) (J Sambrook, E F Frit sch, and T Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York) A subsequence of SEQ ID NO 1 , SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, or SEQ ID NO 21 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides

The nucleotide sequence of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11 , SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, or SEQ ID NO 21, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, or SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, or SEQ ID NO 22, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having endoglucanase activity from strains of different genera or species according to methods well known in the art In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length Even longer probes may be used, e g , nucleic acid probes that are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length Both DNA and RNA probes can be used The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin) Such probes are encompassed by the present invention

A genomic DNA or cDNA library prepared from such other organisms may, therefore, be screened for DNA that hybridizes with the probes descπbed above and encodes a polypeptide having endoglucanase activity Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material In order to identify a clone or DNA that is homologous with the sequences described above, the earner material is used in a Southern blot

For purposes of the present invention, hybridization indicates that the nucleotide sequence hybndizes to a labeled nucleic acid probe corresponding to one of the nucleotide sequences described above under very low to very high stπngency conditions Molecules to that the nucleic acid probe hybridizes under these conditions can be detected using X-ray film

For long probes of at least 100 nucleotides in length, very low to very high stnngency conditions are defined as prehybπdization and hybridization at 42°C in 5X SSPE, 0 3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stπngencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally

For long probes of at least 100 nucleotides in length, the earner material is finally washed three times each for 15 minutes using 2X SSC, 02% SDS preferably at 45°C (very low stringency), more preferably at 50°C (low stringency), more preferably at 55°C (medium stnngency), more preferably at 60⁰C (medium-high stringency), even more preferably at 65⁰C (high stringency), and most preferably at 70⁰C (very high stringency)

For short probes that are about 15 nucleotides to about 70 nucleotides in length, stnngency conditions are defined as prehybndization, hybridization, and washing post- hybndization at about 5°C to about 10⁰C below the calculated T_n, using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48 1390) in 0 9 M NaCI, 0 09 M Tπs-HCI pH 76, 6 mM EDTA, 0 5% NP- 40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0 1 mM ATP, and 0 2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SCC plus 0 1% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 1O⁰C below the calculated T_m Polypeptides Having Biological Activity and Polynucleotides Thereof

Any polypeptide that is poorly secreted or not secreted at all may be used in the methods of the present invention The polypeptide may be any polypeptide having a biological activity of interest The polypeptide having biological activity may be native or heterologous (foreign) to the fungal host cell of interest The term "heterologous polypeptide" is defined herein as a polypeptide that is not native to the host cell, or a native polypeptide in which structural modifications have been made to alter the native polypeptide In a preferred aspect, the polypeptide is an antibody, antigen, antimicrobial peptide, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, or structural protein

In another preferred aspect, the polypeptide is an albumin, collagen, tropoelastin, elastin, or gelatin In a more preferred aspect, the polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase

In an even more preferred aspect, the polypeptide is an alpha-amylase, alpha- 1 ,3-glucanase, alpha-galactosidase, alpha-glucosidase, alpha-1 ,6-mannosιdase, aminopeptidase, amylase, arabinase, beta-agarase beta-amylase, beta-1 ,3-glucanase beta-1,6-glucanase, beta-galactosidase, beta-glucosidase, beta-mannosidase carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, chitosanase, cutinase cyclodextπn glycosyltransferase, deoxyπbonuclease, dextranase, endo-1 ,4-beta- galactanase, endo-1 ,6-beta-galactanase, esterase, fucosidase, glucoamylase, glucocerebrosidase, hyaluronidase, inulinase, invertase, laccase, levanase, licheninase, lipase, lysozyme, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polygalacturonase, polyphenoloxidase, proteolytic enzyme, rhamnogalacturonase, rhamnosidase, ribonuclease, trehalase, transglutaminase, transglycosylase, urokinase, or xylanase

In another even more preferred aspect, the polypeptide is a cellulolylic enzyme or cellulase Examples of cellulolytic enzymes include, but are not limited to endoglucanases, cellobiohydrolases, and beta-glucosidases Other proteins that assist cellulolytic enzyme action are polypeptides having cellulolytic enhancing activity (see, for example, WO 2005/074647, WO 2005/074656, and U S Published Application 2007/0077630) In a most preferred aspect, the polypeptide is an endoglucanase In another most preferred aspect, the polypeptide is a cellobiohydrolase In another most preferred aspect, the polypeptide is a beta-glucosidase In another most preferred aspect, the polypeptide is a polypeptide having cellulolytic enhancing activity

In another more preferred aspect, the polypeptide is a hemicellulase Hemicellulases can be placed into three general categones the endo-acting enzymes that attack internal bonds within the polysaccharide chain, the exo-acting enzymes that act processively from either the reducing or nonreducing end of polysaccharide chain, and the accessory enzymes, acetylesterases and esterases that hydrolyze lignin glycoside bonds, such as coumaric acid esterase and ferulic acid esterase (Wong, K K Y , Tan, L U L , and Saddler, J N , 1988, Multiplicity of β-1,4-xylanase in microorganisms Functions and applications, Microbiol Rev 52 305-317, Tenkanen, M , and Poutanen, K , 1992, Significance of esterases in the degradation of xylans in Xylans and Xyianases, Visser, J , Beldman, G , Kuster-van Someren, M A , and Voragen, A G J , eds , Elsevier, New York, NY, 203-212, Coughlan, M P , and Hazlewood, G P , 1993, Hemicellulose and hemicellulases, Portland, London, UK, Bπgham, J S , Adney, W S , and Mimmel, M E , 1996, Hemicellulases Diversity and applications, in Handbook on Bioethanol Production and Utilization, Wyman, C E , ed , Taylor & Francis, Washington, DC, 119-141)

Hemicellulases include, but are not limited to, xyianases, arabiπofuranosidases, acetyl xylan esterase, glucuronidases, endo-galactanase, mannanases, endo- or exo- arabinases, exo-galactanases, and mixtures thereof Examples of endo-acting hemicellulases and ancillary enzymes include, but are not limited to, endoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase, endoxylanase, and feraxan endoxylanase Examples of exo-acting hemicellulases and ancillary enzymes include, but are not limited to, alpha-L-arabinosidase, beta-L-arabinosidase, alpha-1 ,2-L- fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, beta-D- glucuronidase, beta-D-mannosidase, beta-D-xylosidase, exoglucosidase, exocellobiohydrolase, exomannobiohydrolase, exomannanase, exoxylanase, xylan alpha-glucuronidase, and conifenn beta-glucosidase Examples of esterases include, but are not limited to, acetyl esterases (acetylgalactan esterase, acetylmannan esterase, and acetylxylan esterase) and aryl esterases (coumaric acid esterase and ferulic acid esterase)

In another most preferred aspect, the polypeptide is a xylanase In another most preferred aspect, the polypeptide is a xyloglucanase In another most preferred aspect, the polypeptide is an arabinofuranosidase In another most preferred aspect, the polypeptide is a glucuronidase, e g , alpha-glucuronidase In another most preferred aspect, the polypeptide is an endo-galactanase In another most preferred aspect, the polypeptide is a mannanase In another most preferred aspect, the polypeptide is an endo-arabinase In another most preferred aspect, the polypeptide is an exo-arabinase In another most preferred aspect, the polypeptide is an eπdoarabiπanase In another most preferred aspect, the polypeptide is an endoarabinogalactanase In another most preferred aspect, the polypeptide is an endoglucanase In another most preferred aspect, the polypeptide is an endomannanase In another most preferred aspect, the polypeptide is an endoxylanase In another most preferred aspect, the polypeptide is an feraxan endoxylanase In another most preferred aspect, the polypeptide is an alpha-L- arabinosidase In another most preferred aspect, the polypeptide is an beta-L- arabinosidase In another most preferred aspect, the polypeptide is an beta-glucanase In another most preferred aspect, the polypeptide is an alpha- 1 ,2-L-fucosιdase In another most preferred aspect, the polypeptide is an alρha-D-galactosιdase In another most preferred aspect, the polypeptide is a beta-D-galactosidase In another most preferred aspect, the polypeptide is a beta-D-glucosidase In another most preferred aspect, the polypeptide is a beta-D-glucuronidase In another most preferred aspect, the polypeptide is a beta-D-mannosidase In another most preferred aspect, the polypeptide is a beta-D-xylosidase In another most preferred aspect, the polypeptide is a exoglucosidase In another most preferred aspect, the polypeptide is a exomannobiohydrolase In another most preferred aspect, the polypeptide is a exomannanase In another most preferred aspect, the polypeptide is a exoxylanase In another most preferred aspect, the polypeptide is a xylan alpha-glucuronidase In another most preferred aspect, the polypeptide is a conifeπn beta-glucosidase

In another more preferred aspect, the polypeptide is an esterase As used herein, an "esterase" also known as a carboxylic ester hydrolase, refers to enzymes acting on ester bonds, and includes enzymes classified in EC 3 1 1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (Enzyme Nomenclature, 1992, Academic Press, San Diego, California, with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5, in Eur J Biochem 223 1-5, 1994, Eur J Biochem 232 1-6, 1995, Eur J Biochem 237 1-5, 1996, Eur J Biochem 250 1-6, 1997, and Eur J Biochem 264 610-650, 1999, respectively) Non-limiting examples of esterases include arylesterase, tπacylglycerol lipase, acetylesterase, acetylcholinesterase, cholinesterase, tropinesterase, pectinesterase, sterol esterase, chlorophyllase, L-arabinonolactonase gluconolactonase, uroπolactonase, tannase, retinyl-palmitate esterase, hydroxybutyrate- dimer hydrolase, acylglycerol lipase, 3-oxoadιpate enol-lactonase, 1 ,4-lactonase, galactolipase, 4-pyπdoxolactonase, acylcarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinonolactonase, 6-phosphogluconolactonase, phospholipase A1 , 6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoumaπn lipase, lιmonιn-D-πng-lactonase, steroid-lactonase, tπacetate-lactonase, actinomycm lactonase, orsellinate-depside hydrolase, cephalospoππ-C deacetylase, chlorogeπate hydrolase, alpha-amiπo-acid esterase, 4-methyloxaloacetate esterase, carboxymethylenebutenolidase, deoxyhmonate A-πng-lactonase, 2-acetyl-1-alkylglyceroρhosρhocholιne esterase, fusaπnιne-C ornithinesterase, sinapine esterase, wax-ester hydrolase, phorbol-diester hydrolase, phosphatidylinositol deacylase, sialate O-acetylesterase, acetoxybutynylbithiophene deacetylase, acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase, 2-pyrone-4,6-dιcarboxylate lactonase, N- acetylgalactosaminoglycan deacetylase, juvenile-hormone esterase, bιs(2- ethylhexyl)phthalate esterase, protein-glutamate methylesterase, 11-cιs-retιnyl-palmιtate hydrolase, all-trans-retinyl-palmitate hydrolase, L-rhamnono-1 ,4-lactonase, 5-(3,4- dιacetoxybut-1-ynyl)-2,2'-bιthιoρhene deacetylase, fatty-acyl-ethyl-ester synthase, xylono-1 ,4-lactonase, N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxate benzylesterase, and acetylalkylglycerol acetylhydrolase Esterases that can be used for byconversion of cellulose include acetyl esterases such as acetylgalactan esterase, acetylmannan esterase, and acetylxylan esterase, and esterases that hydrolyze lignin glycoside bonds, such as coumaric acid esterase and ferulic acid esterase

In another most preferred aspect, the polypeptide is an acetyl esterase In another most preferred aspect, the polypeptide is an acetylgalactan esterase In another most preferred aspect, the polypeptide is an acetylmannan esterase In another most preferred aspect, the polypeptide is an acetyl xylan esterase In another most preferred aspect, the polypeptide is an aryl esterase In another most preferred aspect, the polypeptide is a coumaric acid esterase In another most preferred aspect, the polypeptide is a ferulic acid esterase

In another more preferred aspect, the polypeptide is a lipase In another more preferred aspect, the polypeptide is a phospholipase, e g , phospholipase A1, phospholipase A2, phospholipase C, phospholipase C, or phospholipase D In another more preferred aspect, the polypeptide is a cutinase In another most preferred aspect, the polypeptide is a glucose isomerase In another most preferred aspect, the polypeptide is a xylose isomerase In another more preferred aspect, the polypeptide is a proteolytic enzyme

Proteases are well known in the art and refer to enzymes that catalyze the cleavage of peptide bonds

In another most preferred aspect, the polypeptide is a serine protease In another most preferred aspect, the polypeptide is a metalloprotease In another most preferred aspect, the polypeptide is a thiol protease

In another more preferred aspect, the polypeptide is a peptidase In another most preferred aspect, the polypeptide is an aminopeptidase, e g . dipeptidylamiπopeptidase or tπpeptidylaminopeptidase In another most preferred aspect, the polypeptide is a carboxypeptidase

In another more preferred aspect, the polypeptide is a laccase In another more preferred aspect, the polypeptide is a peroxidase In another more preferred aspect, the polypeptide is a starch degrading enzyme

In another most preferred aspect, the polypeptide is an alpha-amylase In another most preferred aspect, the polypeptide is an amyloglucosidase In another most preferred aspect, the polypeptide is pullulanase In another most preferred aspect, the polypeptide is a debranching enzyme In another most preferred aspect, the polypeptide is a cylcodextrin glycosyltransferase

A polynucleotide encoding a catalytic domain, mature polypeptide, or full-length polypeptide of a polypeptide having biological activity, or a portion thereof, may be obtained from any organism

A polynucleotide encoding a polypeptide having biological activity may be obtained from a gene encoding a bacterial polypeptide For example, the polypeptide may be a Gram positive bacterial polypeptide including, but not limited to, a Bacillus,

Streptococcus, Streptomyces, Staphylococcus, Entero∞ccus, Lactobacillus,

Lacto∞ccus, Clostridium, Geobacillus, or Oceanobaciilυs polypeptide, or a Gram negative bacterial polypeptide including, but not limited to, an E coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobactenum, Fusobactenum, llyobacter

Neisseria, or Ureaplasma polypeptide

In a preferred aspect, a polynucleotide encoding a polypeptide having biological activity may be obtained from a gene encoding a Bacillus alkalophilus, Bacillus amyloliquefaαens, Bacillus brevis, Bacillus αrculans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus taut us, Bacillus lentus, Bacillus hcheniformis, Bacillus megateπum, Bacillus pumitus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thunngiensis polypeptide

In another preferred aspect, a polynucleotide encoding a polypeptide having biological activity may be obtained from a gene encoding a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus ubens, or Streptococcus equi subsp Zooepidemicus polypeptide

In another preferred aspect, a polynucleotide encoding a polypeptide having biological activity may be obtained from a gene encoding a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces grtseus, or Streptomyces lividans polypeptide

A polynucleotide encoding a polypeptide having biological activity may also be obtained from a gene encoding a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schtzosaccharomyces, or Yarrowia polypeptide, or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaπcus, Atternana, Aspergillus, Aureobasidium, Botryospaeria, Ceπpoπopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobotus, Coprinopsis, Coptotermes, Corynascus, Cryphonectπa, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaena, Magnaporthe, Melanocarpus, Menpilus, Mucor, Mycehophthora, Neocallimastix, Neurospora, Paecilomyces, Peniαllium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania Pseudotnchonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoaseus, Thielavia, Totypodadium, Trichoderma, Trichophaea, Vertiαlhum, Volvaπella, or Xylaria polypeptide

In a preferred aspect, a polynucleotide encoding a polypeptide having biological activity may be obtained from a gene encoding a Saccharomyces cartsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasu, Saccharomyces kluyveπ, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide

In another preferred aspect, a polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosponum keratinophilum, Chrysosponum lucknowense, Chrysosponum tmpicum, Chrysosponum merdarium, Chrysosporium mops, Chrysosponum pannicola, Chrysosponum queenslandicum, Chrysosponum zonatum, Cladorrhinum foecundissimum, Fusarium bactndioides, Fusarium cerealis, Fusanum crookwellense, Fusarium culmorum, Fusarium graminearum, Fusanum graminum, Fusanum heterosporum, Fusanum negundi, Fusanum oxysporum, Fusarium reticulatum, Fusanum roseum, Fusarium sambuαnum, Fusanum sarcochroum, Fusanum sporotnchioides, Fusanum sulphureum, Fusanum torulosum, Fusanum trichothecioides, Fusarium venenatum, Humicola gnsea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillmm funiculosum, Pemαllium purpurogenum, Phanerochaete chrysosponum, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspore, Thielavia ovispora, Thielavia peruviana, Thielavia spededomum, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Thielavia vanospora, Thielavia wareingii, Tnchoderma harzianum, Trichoderma kontngii, Trichoderma longibrachiatum Tnchoderma reesei, or Tnchoderma vinde In a particularly preferred aspect, the polypeptide having biological activity is a beta-glucosidase Examples of beta-glucosidases that can be used as sources for the polynucleotides in the methods of the present invention, include, but are not limited to, an Aspergillus oryzae bela-glucosidase (WO 02/095014, WO 04/099228), Aspergillus 5 aculeatus beta-glucosidase (Kawaguchi et al , 1996, Gene 173 287-288), Aspergillus avenaceus (GENBANK™ accession no AY943971), Aspergillus fumigatus (GENBANK™ accession no XM745234), Aspergillus kawacfw (GENBANK™ accession no AB003470), Aspergillus niger (GENBANK™ AJ132386), Magnaporthe grisea (GENBANK™ accession no AY849670), Phanerochaete chrysosponum (GENBANK™

10 accession no AB253327), Talaromyces emersonu (GENBANK™ accession no AY072918), and Trichoderma reesei (GENBANK™ accession nos U09580, AB003110, AY281374, AY281375, AY281377, AY281378, and AY281379) Variants Of beta- glucosidases may also be used as sources for the polynucleotides such as those described in WO 04/099228

I₅ In a preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a Family 1 beta-glucosidase gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a

20 polynucleotide obtained from a Family 3 beta-glucosidase gene

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of

25 the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene comprising SEQ ID NO 23 that encodes the polypeptide of SEQ ID NO 24 or an Aspergillus oryzae beta-glucosidase mutant gene comprising SEQ ID NO 25 that encodes the polypeptide of SEQ ID NO 26

In another preferred aspect, the full-length polypeptide, mature polypeptide, or

30 catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene compπsing SEQ ID NO

35 27 that encodes the polypeptide of SEQ ID NO 28

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a Peπicilhum brasilianum strain IBT 20888 beta- glucosidase gene In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a Penicillium brasilianum strain IBT 20888 beta- glucosidase gene comprising SEQ ID NO 29 that encodes the polypeptide of SEQ ID NO 30

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a Tnchoderma reesei strain No QM9414 beta-glucosidase gene In another most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a Tnchoderma reesei strain No QM9414 beta-glucosidase gene compπsing SEQ ID NO 31 that encodes the polypeptide of SEQ ID NO 32 (GENBANK™ accession no U09580) In another preferred aspect, the beta-glucosidase is naturally secreted In another preferred aspect, the beta-glucosidase is not naturally secreted

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide obtained from a gene encoding a homologous polypeptide comprising an amino acid sequence that has a degree of identity to the amino acid sequences of the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, or SEQ ID NO 32 of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably 96%, 97%, 98%, or 99%, which have endoglucanase activity In a preferred aspect, the homologous polypeptide has an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, or SEQ ID NO 32

In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase, or a portion thereof, is encoded by a polynucleotide that hybridizes under very low stringency conditions, preferably low stnngency conditions, more preferably medium stnngency conditions, more preferably medium-high stnngency conditions, even more preferably high stringency conditions, and most preferably very high stnngency conditions with (ι) SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO" 29, or SEQ ID NO" 31 , (ιι) the cDNA sequence contained in SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, or SEQ ID NO 31, (in) a subsequence of (ι) or (n), or (ιv) a complementary strand of (ι), (n), or (ιιi) (J Sambrook, E F Fπtsch, and T Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York) A subsequence of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, or SEQ ID NO 31 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides

The nucleotide sequence of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27 SEQ ID NO 29, or SEQ ID NO 31 , or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, or SEQ ID NO 32, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having beta-glucosidase activity from strains of different genera or species according to methods well known in the art In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length Even longer probes may be used, e g , nucleic acid probes that are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length Both DNA and RNA probes can be used The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin) Such probes are encompassed by the present invention

Signal Peptides The signal peptide can be any appropriate signal peptide recognized by a host cell for extracellular secretion of a fusion protein of the present invention The signal sequence is preferably that which is naturally associated with the endoglucanase component of the fusion protein to be expressed

The 5' end of the coding sequence of the nucleotide sequence encoding a polypeptide may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide However, any signal peptide coding region that directs the expressed fusion protein into the secretory pathway of a host cell of choice, i e , secreted into a culture medium, may be used in the present invention

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequence obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase, Tnchoderma reesei cellobiohydrolase I, Tnchoderma reesei cellobiohydrolase II, Tnchoderma reesei endoglucanase I, Tnchoderma reesei endoglucanase II, Tnchoderma reesei endoglucanase III, Tnchoderma reesei endoglucanase IV, Tnchoderma reesei endoglucanase V, Tnchoderma reesei xylanase I, Tnchoderma reesei xylanase II, and Tnchoderma reesei beta-xylosidase

Useful signal peptide coding sequences for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase Other useful signal peptide coding sequence are described by Romanos et al , 1992, supra

In a preferred aspect, the signal peptide coding sequence is obtained from a gene that encodes an endoglucanase In a more preferred aspect, the signal peptide coding sequence is obtained from a gene that encodes an endoglucanase V In an even more preferred aspect, the signal peptide coding sequence is obtained from a Humicola insolens gene that encodes an endoglucanase V in a most preferred aspect, the signal peptide coding sequence encodes amino acids 1 to 21 of SEQ ID NO 2 In another most preferred aspect, the signal peptide coding sequence is nucleotides 1 to 63 Of SEQ ID NO 1 The fusion protein may further comprise a second signal peptide that is associated with the beta-glucosidase component of the fusion protein The signal peptide coding sequence may be the signal peptide coding sequence that is naturally associated with the coding sequence of the polypeptide having biological activity or may be a different signal peptide coding sequence such as one of those described above In a preferred aspect, the signal peptide coding sequence is a sequence naturally associated with a beta-glucosidase coding sequence Linkers

As mentioned supra, many endoglucanases have a multidomain structure consisting of a catalytic domain separated from one or more carbohydrate binding modules by a linker peptιde(s) In the methods of the present invention, the fusion protein constructs can further comprise a linker coding sequence located 3' to the sequence comprising the endoglucanase catalytic domain and 5' to the sequence compπsing the catalytic domain of the polypeptide having biological activity

The linker can be obtained from the same gene as the catalytic domain of the endoglucanase or from a different endoglucanase gene On the other hand, the linker can be synthetic in origin

Examples of linkers that can be used in the methods of the present invention include, but are not limited to, linkers obtained from the genes for the Trichoderma reesei cellobiohydrolase I (Srisodsuk et al , 1Θ93, Journal of Biological Chemistry 268 20765-20761), Hypocrea jeconna (formerly Trichoderma reesei) Cel7A cellobiohydrolase (Mulakala et al , 2005, Proteins 60 598-605), Humicola insolens endoglucanase V, and Thielavia terrestris NRRL 8126 CEL7C endoglucanase

In a preferred aspect, the linker is obtained from a Humicola insolens endoglucanase gene In another preferred aspect, the linker is obtained from a Trichoderma reesei endoglucanase gene In a more preferred aspect, the linker is obtained from a Humicola insolens endoglucanase V (eg5) gene

In another preferred aspect, the linker is obtained from a Thielavia terrestris endoglucanase gene In another more preferred aspect, the linker is obtained from a Thielavia terrestns NRRL 8126 CEL7C endoglucanase gene

In a preferred aspect, the linker is at least 5 amino acid residues In a more preferred aspect, the linker is at least 15 amino acid residues In a most preferred aspect, the linker is at least 25 amino acid residues

In a preferred aspect, the linker is between about 5 to about 60 amino acid residues In a more preferred aspect, the linker is between about 15 to about 50 amino acid residues In a most preferred aspect, the linker is between about 25 to about 45 amino acid residues

Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are defined as contiguous amino acid sequences with a discrete fold having carbohydrate binding activity, and are commonly found within carbohydrate-active enzymes A number of types of CBMs have been described, and a majority thereof bind to insoluble polysacchandes (see Boraston et at,

2004, BlOChem J 382 769-781)

Carbohydrate binding modules have been characterized which mediate interaction with, for example, crystalline cellulose, non-crystalline cellulose, chitin, beta-1 ,3-glucans and beta-1 ,3-1,4-mιxed linkage glucans, xylan, mannan, galactan and starch Carbohydrate binding modules, occur in frequently in multi-domain cellulases While some CBMS confer specific binding to a subset of carbohydrate structures, others are more general in their ability to associate with vanous polysacchandes CBMs which confer binding to cellulose are sometimes referred to as cellulose binding domains, or CBDs (Boraston et al, 2004, Biochem J 382 769-781) CBMs are grouped by amino acid similarity, currently, 48 CBM families are described

Glycoside hydrolases can comprise more than one catalytic domain and one, two, three, or more CBMs, and optionally further comprise one or more polypeptide amino acid sequence regions linking the CBM(s) with the catalytic domaιn(s), a region of the latter type usually being denoted a "linker" Examples of hydrolytic enzymes compnsing a CBM are cellulases, xylanases, mannanases, arabinofuranosidases acetylesterases and chitinases (See P Tomme ef a/ , Cellulose-Binding Domains - Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates, John N Saddler and Michael H Penner (Eds ), ACS Symposium Series, No 618, 1996) A CBM may be located at the N or C terminus or at an internal position of a protein or polypeptide The region of a polypeptide or protein that constitutes a CBM typically consists of more than about 30 and less than about 250 ammo acid residues For example those CBMs listed and classified in Family I consist of 33-37 amino acid residues, those listed and classified in Family 2a consist of 95-108 amino acid residues, and those listed and classified in Family 6 consist of 85-92 amino acid residues Accordingly, the molecular weight of an amino acid sequence constituting a CBM will typically be in the range of from about 4 kDa to about 40 kDa, and usually below about 35 kDa

In the methods of the present invention, any CBM may be used The CBM may be naturally associated with the endoglucanase or may be foreign to the endoglucanase

In a preferred aspect, a CBM is obtained from a Tnchoderma reesei eπdoglucanase (EG) gene In a more preferred aspect, a CBM is obtained from a Tπchoderma reesei endoglucanase EGI gene In another more preferred aspect, a CBM is obtained from a Tπchoderma reesei endoglucanase EGII gene In another more preferred aspect, a CBM is obtained from a Tnchoderma reesei endoglucanase EGV In another preferred aspect, a CBM is obtained from a Tπchoderma reesei cellobiohydrolase (CBH) gene In another preferred aspect, a CBM is obtained from a Tnchoderma reesei CBHI gene (Tern ef a/ , 1987, Gene 51 42-52, Linder and Teen, 1996, Biochemistry 93 12251-12255) In another preferred aspect, a CBM is obtained from a Tnchoderma reesei CBHII gene In another preferred aspect, a CBM is obtained from a Thielavia terrestris endoglucanase gene In another more preferred aspect, a CBM is obtained from a Thielavia terrestns NRRL 8126 CEL7C endoglucanase gene

Cleavage Site In the methods of the present invention, the fusion protein constructs can further compnse a nucleotide sequence encoding a cleavage site The cleavage site is preferably located between the sequence compnsing at least the endoglucanase catalytic domain and the sequence comprising at least the catalytic domain of the polypeptide having biological activity Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide having biological activity from the fusion protein

Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et a/ , 2003, J lnd Microbiol Biotechnol 3 568- 76, Svetina ef a/ , 2000, J Biotechnol 76 245-251, Rasmussen-Wilson ef a/ , 1997, Appl Environ Microbiol 63 3488-3493, Ward et al , 1995, Biotechnology 13 498-503, and Contreras ef al , 1991 , Biotechnology 9 378-381), an Me-(GIu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton ef al , 1986, Biochem 25 505-512), a Asp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after the lysine (Colhns-Racie ef al , 1995, Biotechnology 13 982-987), a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter ef al , 1989, Proteins Structure, Function, and Genetics 6 240-248), a Leu-Val-Pro-Arg-Gly-Ser site which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4 35- 48J, a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease after the GIn (Stevens, 2003, supra), and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the GIn (Stevens, 2003, supra)

Fusion Proteins A fusion protein having biological activity of the present invention comprising a signal peptide, at least the catalytic domain of an endoglucanase or a portion thereof, and at least the catalytic domain of a polypeptide having biological activity or a portion thereof, increases secretion of the fusion protein compared to the absence of at least the catalytic domain of the endoglucanase or a portion thereof In each of the preferred aspects below, the components of a fusion protein are linked in frame from the N terminus to the C terminus of the protein

In a preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, and a mature polypeptide of an endoglucanase In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, and a full-length polypeptide of a polypeptide having biological activity In another preferred aspect, the fusion protein comprises a signal peptide, a full- length polypeptide of a polypeptide having biological activity, and a catalytic domain of an endoglucanase In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, and a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein compπses a signal peptide, a full- length polypeptide of a polypeptide having biological activity, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a full-length polypeptide of an endoglucanase (signal peptide and mature polypeptide), and a full- length polypeptide of a polypeptide having biological activity In another preferred aspect, the fusion protein comprises a full-length polypeptide of a polypeptide having biological activity and a full-length polypeptide of an endoglucanase (signal peptide and mature polypeptide)

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a linker and/or a carbohydrate binding module, and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a linker and/or a carbohydrate binding module, and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, and a mature polypeptide of an endoglucanase In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a linker and/or a carbohydrate binding module, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a linker and/or a carbohydrate binding module, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a linker and/or a carbohydrate binding module, and a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein compπses a signal peptide, a full- length polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a linker and/or a carbohydrate binding module, and a full-length polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein compπses a signal peptide, a full- length polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a second signal peptide, and a catalytic domain of a polypeptide having biological activity In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a second signal peptide, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a second signal peptide, and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a second signal peptide, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a second signal peptide, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, a second signal peptide, and a catalytic domain of an endoglucanase In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a second signal peptide, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, a second signal peptide, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a linker and/or a carbohydrate binding module, a second signal peptide; and a catalytic domain of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, a second signal peptide, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a linker and/or a carbohydrate binding module, a second signal peptide, and a catalytic domain of a polypeptide having biological activity In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, a second signal peptide, and a mature polypeptide of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a catalytic domain of an endoglucanase, a linker and/or a carbohydrate binding module, a second signal peptide, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, a second signal peptide, and a catalytic domain of an endoglucanase

In another preferred aspect, the fusion protein comprises a signal peptide, a mature polypeptide of an endoglucanase, a linker and/or a carbohydrate binding module, a second signal peptide, and a mature polypeptide of a polypeptide having biological activity

In another preferred aspect, the fusion protein comprises a signal peptide, and a mature polypeptide of a polypeptide having biological activity, a linker and/or a carbohydrate binding module, a second signal peptide, and a mature polypeptide of an endoglucanase In another preferred aspect, for each of the preferred aspects above, the fusion protein may alternatively comprise a portion of the catalytic domain, the mature polypeptide, or the full-length polypeptide of an endoglucanase or a polypeptide having biological activity The a portion of the endoglucanase may or may not have endoglucanase activity In a more preferred aspect, the portion of the endoglucanase has endoglucanase activity Promoters

The promoter region can be any appropriate promoter sequence recognized by a host cell for expression of a fusion protein The promoter sequence contains transcπptional control sequences that mediate the expression of the polypeptide The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, tandem, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either native or foreign (heterologous) to the host cell Exemplary promoters include both constitutive promoters and inducible promoters Examples of suitable promoters for directing transcription of the fusion protein constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E coll lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al , 1978, Proceedings of the National Academy of Sciences USA 75 3727-3731), as well as the tac promoter (DeBoer ef a/ , 1983, Proceedings of the National Academy of Sciences USA 80 21-25) Further promoters are descπbed in "Useful proteins from recombinant bacteria" in Scientific Amencan, 1980, 242 74-94, and in Sambrook et al , 1989, supra

Examples of suitable promoters for directing transcnption of the fusion protein constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamon glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusanum venenatum amyloglucosidase (WO 00/56900), Fusanum venenatum Dana (WO 00/56900), Fusanum venenatum Quinn (WO 00/56900), Fusanum oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Tnchoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Tnchoderma reesei endoglucanase III, Tnchoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Tnchoderma reesei xylanase I₁ Tnchoderma reesei xylanase II, Tnchoderma reesei beta-xylosidase, Coprinus cinereus beta-tubulin and Trichoderma reesei swollenin, as well as the NA2-tpι promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3- phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are described by Romanos et al , 1992, Yeast a 423-488 In a preferred aspect, the promoter is a cellobiohydrolase promoter In a more preferred aspect, the promoter is a cellobiohydrolase I (cbh1) promoter In an even more preferred aspect, the promoter is a Trichoderma reesei cellobiohydrolase I gene (cbh1) promoter In a most preferred aspect, the promoter is the Tπchoderma reesei CbM promoter of nucleotides 505 to 1501 of SEQ ID NO 29 (GENBANK™ accession no D86235) In another more preferred aspect, the promoter is a cellobiohydrolase Il (cbh2) promoter In another even more preferred aspect, the promoter is a Tnchoderma reesei cellobiohydrolase Il gene (cbh2) promoter In another most preferred aspect, the promoter is the Tnchoderma reesei cbh2 promoter of nucleotides 1 to 582 of SEQ ID NO 30 (GENBANK™ accession no M55080) In another preferred aspect, the promoter is the NA2-tpι promoter In another preferred aspect, the promoter is a TAKA amylase promoter In another preferred aspect, the promoter is a Fusarium venenatum amyloglucosidase promoter In another preferred aspect, the promoter is a Fusarium oxysporum trypsin-like protease promoter In another preferred aspect, the promoter is an Aspergillus niger or Aspergillus awamon glucoamylase (glaA) promoter

In a preferred aspect, the promoter region drives expression of the first, second, and third polynucleotides, and alternatively also the fourth polynucleotide

Terminators The terminator can be any suitable transcription terminator sequence recognized by a host cell to terminate transcription The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide Any terminator that is functional in the host cell of choice may be used in the present invention

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusaπum oxysporum trypsin-like protease Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-ρhosρhate dehydrogenase Other useful terminators for yeast host cells are described by Romanos ef a/ , 1992, supra In a preferred aspect, the terminator is a cellobiohydrolase gene terminator In a more preferred aspect, the terminator is a cellobiohydrolase I gene (cbM) terminator In an even more preferred aspect, the terminator is a Tnchoderma reesei cellobiohydrolase I gene (cbM) terminator In a most preferred aspect, the terminator is the Tnchoderma reesei cbM terminator of SEQ ID NO 31 In another more preferred aspect, the terminator is a cellobiohydrolase Il gene (cbh2) terminator In another even more preferred aspect, the terminator is a Tnchoderma reesei cellobiohydrolase Il gene (cbh2) terminator In another most preferred aspect, the terminator is the Tnchoderma reesei cbh2 terminator of SEQ ID NO 32

In another preferred aspect, the terminator is a TAKA amylase terminator In another preferred aspect, the promoter is a Fusaπum oxysporum trypsin-like protease terminator In another preferred aspect, the promoter is an Aspergillus niger or Aspergillus awamon glucoamylase iglaA) terminator

Other Regulatory Sequences The fusion protein constructs can further comprise other regulatory elements such as a leader, polyadenylation sequence, and other elements

The regulatory sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding a fusion protein Any leader sequence that is functional in the host cell of choice may be used in the present invention

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans tnose phosphate isomerase Suitable leaders for yeast host cells are obtained from the genes for

Saccharomyces cerevisiae enolase (ENO-1) Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) The regulatory sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA Any polyadeπylatioπ sequence that is functional in the host cell of choice may be used in the present invention

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin- like protease, and Aspergillus niger alpha-glucosidase

Useful polyadenylation sequences for yeast host cells are descnbed by Guo and Sherman, 1995, Molecular Cellular 8/o/ogy 15 5983-5990

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems In yeast, the ADH2 system or GAL1 system may be used In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences Other examples of regulatory sequences are those that allow for gene amplification In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate and the metallothionein genes which are amplified with heavy metals In these cases the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence

The fusion protein constructs preferably contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like

Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin chloramphenicol, or tetracycline resistance Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3 Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase) argB (ornithine carbamoyltransferase), bar (phosphinothπαn acetyltransferase), hph (hygromyciπ phosphotransferase), niaD (nitrate reductase), pyrG (orotιdιne-5'- phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a fusion protein, a promoter, and transcriptional and translational stop signals The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites Alternatively, a polynucleotide encoding a fusion protein may be expressed by inserting the nucleotide sequence or a fusion protein construct comprising the sequence into an appropriate vector for expression In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression

The recombinant expression vector may be any vector (e g , a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced The vectors may be linear or closed circular plasmids

The vector may be an autonomously replicating vector, / e , a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, eg , a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome The vector may contain any means for assunng self-replication Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used

The vectors of the present invention preferably contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells Examples of bacterial, yeast, filamentous fungal selectable markers are descπbed herein

A vector of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise locatιon(s) in the chromosome(s) To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to enhance the probability of homologous recombination The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell Furthermore the integrational elements may be non-encoding or encoding nucleotide sequences On the other hand the vector may be integrated into the genome of the host cell by non-homologous recombination

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

Examples of bacterial ongins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E coll, and ρUB110, ρE194 pTAiOδO, and pAMβi permitting replication in Bacillus

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4 the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et a/ , 1991, Gene 98 61-67, Cullen et al , 1987, Nucleic Acids Research 15 9163-9175, WO 00/24883) Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883

More than one copy of a polynucleotide encoding a fusion protein may be inserted into the host cell to increase production of the gene product An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropπate selectable agent

The procedures used to hgate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e g , Sambrook et a/ , 1989, supra)

Host Cells

The present invention also relates to recombinant fungal host cells, comprising a polynucleotide encoding a fusion protein of the present invention, which are advantageously used in the recombinant production of the protein A vector comprising a polynucleotide of the present invention is introduced into a fungal host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication

The fungal host cell may be any fungal cell useful in the recombinant production of a polypeptide of the present invention

"Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycete (as defined by Hawksworth et al , In, Ainsworth and

Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press,

Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al , 1995, supra, page 171) and all mitospoπc fungi (Hawksworth et al , 1995, supra)

In a preferred aspect, the fungal host cell is a yeast cell "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes) Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as descπbed in Biology and Activities of Yeast (Skinner, F A , Passmore, S M , and Davenport, R R , eds, Soc App Bacteπol Symposium Series No 9, 1980) In a more preferred aspect, the yeast host cell is a Candida, Hansenula,

Ktuyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell

In a most preferred aspect, the yeast host cell is a Saccharomyces cartsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces ovifoimis cell In another most preferred aspect, the yeast host cell is a Ktuyvemmyces lactis cell In another most preferred aspect, the yeast host cell is a Yarrowia lipolytics cell

In another preferred aspect, the fungal host cell is a filamentous fungal cell

"Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth ef a/ , 1995, supra) The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysacchandes Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative

In a more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceπpoπopsis, Chrysosporium, Coprinus, Conolus, Cryptococcus, Fihbasidium, Fusanum, Humi∞la, Magnaporthe, Mucor, Myceliophthora, Neocallimasύx, Neurospora, Paeαlomyces, Penαllium, Phanerochaete, Phtebia, Piromyoes, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Tnchoderma cell In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamon, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell In another most preferred aspect, the filamentous fungal host cell is a Fusanum bactπdioides, Fusarium cerealis, Fusanum crookwellense, Fusanum culmorum, Fusanum grammearum, Fusanum graminum, Fusarium heterosporum, Fusanum negundi, Fusanum oxysporum, Fusanum reticulatum, Fusanum roseum, Fusanum sambuαnum, Fusanum sar∞chroum, Fusarium sporotnchioides, Fusanum sulphureum, Fusanum torulosum, Fusanum tnchotheαoides, or Fusarium venenatum cell In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adust a, Cenporiopsis anemna, Cenporiopsis anemna, Cenponopsis caregiea, Cenporiopsis gilvescens, Cenporiopsis pannoαnta, Cenponopsis nvulosa Cenporiopsis subrufa, Cenponopsis subvermispora, Chrysosporium keratmophilum, Chrysosporium lucknowense, Chrysosporium troμcum, Chrysosponum merdanum, Chrysosporium mops, Chrysosponum pannicola, Chrysosporium queenslandicum, Chrysosponum zonatum, Coprinus αnereus, Conolus hirsutus, Humicola msolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Pemαllium purpurogenum, Phanerochaete chrysosponum, Phlebia radiata, Pleurotus eryngii, Thielavia terrestns, Trametes villosa, Trametes versicolor, Trichodenva harzianum, Tnchodenna konmgii, Tnchoderma longibrachiatum, Tnchoderma reesei, or Trichodenva vinde cell Fungal cells may be transformed by a process involving protoplast formation transformation of the protoplasts, and regeneration of the cell wall in a manner known per se Suitable procedures for transformation of Aspergillus and Tnchoderma host cells are described in EP 238 023 and Yelton et al , 1984, Proceedings of the National Academy of Sciences USA 81 1470-1474 Suitable methods for transforming Fusanum species are described by Malardier et al , 1989, Gene 78 147-156, and WO 96/00787 Yeast may be transformed using the procedures descnbed by Becker and Guarente, In Abelson, J N and Simon, M I , editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Iπc , New York, lto et al , 1983, Journal of Bacteriology 153 163, and Hinnen ef al , 1978, Proceedings of the National Academy of Sciences USA 75 1920

Production and Recovery

In the methods of the present invention, the fungal host cell is cultivated in a nutπent medium suitable for production of a polypeptide having biological activity using methods well known in the art For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industnal fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated The cultivation takes place in a suitable nutrient medium compnsing carbon and nitrogen sources and inorganic salts, using procedures known in the art Suitable media are available from commercial suppliers or may be prepared according to published compositions (e g , in catalogues of the Ameπcan Type Culture Collection)

In the methods of the present invention, the polypeptide having biological activity is selected from the group consisting of a fusion protein, components of the fusion protein, and a combination of the fusion protein and the components thereof

In a preferred aspect, the polypeptide having biological activity is a fusion protein

In another preferred aspect, the polypeptide having biological activity is a component(s) of a fusion protein

In another preferred aspect, the polypeptide having biological activity is a combination of a fusion protein and components thereof The polypeptides having biological activity may be detected using methods known in the art that are specific for the polypeptides These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate For example, an enzyme assay may be used to determine the activity of the polypeptide, as described herein, which can include both endoglucanase activity and a specific biological activity

The resulting polypeptide having biological activity, e g beta-glucosidase fusion protein or a component thereof, may be recovered using methods known in the art For example, the polypeptide may be recovered from the nutπent medium by conventional procedures including, but not limited to, centnfugation, filtration, extraction, spray-drying, evaporation, or precipitation

The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e g , ion exchange, affinity, hydrophobic, chromatofocusiπg, and size exclusion), electrophoretic procedures (e g , preparative isoelectric focusing), differential solubility (e g , ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e g , Protein Purification, J -C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides

Compositions

The present invention also relates to compositions comprising a polypeptide having biological activity of the present invention Preferably, the compositions are enriched in such a polypeptide The term "enriched" indicates that the biological activity of the composition has been increased, e g , with an enrichment factor of at least 1 1

The composition may comprise a polypeptide of the present invention as the major enzymatic component, e g , a mono-component composition Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextnn glycosyltransferase, deoxyπbonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutammase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme ribonuclease, transglutaminase, or xylanase The additional enzyme(s) may be produced, for example, by a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamoπ, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae, Fusanum, preferably Fusaπum bactndioides, Fusarium cerealis, Fusaπum crookwellense, Fusarium culmorum, Fusarium graminearum, Fusanum graminum, Fusanum heterosporum, Fusaπum negundi, Fusanum oxysporum, Fusaπum reticulatum, Fusanum roseum, Fusanum sambucinum, Fusanum sarcochroum, Fusanum sulphureum, Fusanum toruloseum, Fusanum tnchotheαoides, or Fusanum venenatum, Humicola, preferably Humicola tnsolens or Humicola lanuginosa, or Tπchoderma, preferably Tnchoderma harztanum, Trichoderma konmgii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viπde

The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition For instance, the polypeptide composition may be in the form of a granulate or a microgranulate The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art

Examples are given below of preferred uses of the polypeptide compositions of the invention The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art

5 Uses

The present invention is also directed to methods for using the fusion proteins or components thereof, or compositions thereof

Methods of Processing Cellulosic Material

10 The methods of the present invention are particularly useful for improving the secretion of polypeptides having cellulolytic or hemicellulolytic activity in commercially important quantities, which can be used to degrade or convert lignocellulosic material Such polypeptides include, but are not limited to endoglucanases, cellobiohydrolases, beta-glucosidases, xylanases, beta-xylosidases, arabinofuranosidases, acetyl xylan

I₅ esterases, and ferulic acid esterases Consequently, the present invention also relates to methods for degrading or converting a cellulosic material, compπsing treating the cellulosic material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a fusion protein or a component thereof having cellulolytic activity or hemicellulolytic activity obtained according to the instant methods

20 For purposes of illustration, a polypeptide having beta-glucosidase activity obtained according to the methods of the present invention, e g , a beta-glucosidase fusion protein or a component thereof, is used for illustrative purposes

Cellulosic biomass can include, but is not limited to, wood resources, municipal solid waste, wastepaper, crops, and crop residues (see, for example, Wiselogel ef a/ ,

25 1995, in Handbook on Bioethanol (Charles E Wyman, editor), pp 105-118, Taylor & Francis, Washington D C , Wyman, 1994, Btoresource Technology 50 3-16, Lynd, 1990, Applied Biochemistry and Biotechnology 24/25 695-719, Mosier et at , 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T Scheper, managing editor, Volume 65, pp 23-40,

30 Springer-Verlag, New York)

The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymenc lignin covalently cross-linked to hemicellulose Cellulose is a

35 homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matπx It will be understood herein that the term "cellulosic material" or "cellulose" also encompasses lignocellulose

In the methods of the present invention, the cellulolytic enzyme composition may compπse any protein involved in the processing of cellulosic matenal to glucose, or hemicellulose to xylose, mannose, galactose, and arabinose, their polymers, or products derived from them as described below The cellulolytic enzyme composition may be a monocomponent preparation, e g , an endoglucanase a multicomponent preparation, e g , endoglucanase, cellobiohydrolase, beta-glucosidase, or a combination of multicomponent and monocomponent protein preparations The cellulolytic proteins may have activity, / e , hydrolyze cellulose, either in the acid, neutral, or alkaline pH- range The cellulolytic enzyme composition can further compπse a polypeptide having cellulolytic enhancing activity according to WO 2005/074647, WO 2005/074656, and U S Published Application 2007/0077630

The cellulolytic protein may be of fungal or bacterial oπgin, which may be obtainable or isolated and purified from microorganisms that are known capable of producing cellulolytic enzymes, e g , species of Bacillus, Pseudomonas, Humicola, Coprinus, Thielavia Fusanum, Myceliophthora, Acremonium, Cephalospoπum Scytalidmm, Peniαlhum or Aspergillus (see, for example, EP 458162), especially those produced by a strain selected from Humicola insolens (reclassified as Scytalidmm thermophilum, see for example, U S Patent No 4,435,307), Coprinus αnereus, Fusanum oxysporum, Myceliophthora theimophila, Menpilus giganteus, Thielavia terrestris, Acremonium sp , Acremonium persiαnum, Acremonium acremonium, Acremonium brachypeniutn, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pmkertoniae, Acremonium roseognseum, Acremonium incoloratum, and Acremonium furatum, preferably from Humicola insolens DSM 1800, Fusanum oxysporum DSM 2672, Myceliophthora theimophila CBS 117 65, Cephalosponum sp RYM-202, Acremonium sp CBS 47894, Acremonium sp CBS 265 95, Acremonium persiαnum CBS 169 65, Acremonium acremonium AHU 9519, Cephalosponum sp CBS 535 71 , Acremonium brachypenium CBS 866 73, Acremonium dichromosporum CBS 683 73, Acremonium obclavatum CBS 311 74, Acremonium pmkertoniae CBS 157 70, Acremonium roseognseum CBS 134 56, Acremonium incoloratum CBS 146 62, and Acremonium furatum CBS 2997OH Cellulolytic proteins may also be obtained from Trichoderma (particularly Tnchoderma vinde, Trichoderma reesei, and Tnchoderma koningu), alkalophilic Bacillus (see, for example, U S Patent No 3,844,890 and EP 458162), and Streptomyces (see, for example, EP 458162) Chemically modified or protein engineered mutants of cellulolytic proteins may also be used

Especially suitable cellulolytic proteins are the cellulases described in EP 495,257, EP 531 ,372, WO 96/11262, WO 96/29397, WO 98/08940 Other examples are cellulase variants such as those descnbed in WO 94/07998, EP 531 ,315, U S Patent No 4,435,307, U S Patent No 5,457,046, U S Patent No 5,648,263, U S Patent No 5,686,593, U S Patent No 5,691 ,178, U S Patent No 5,763,254, U S Patent No 5,776,757, WO 89/09259, WO 95/24471 , WO 98/12307, and PCT/DK98/00299 As mentioned above, the cellulolytic proteins used in the methods of the present invention may be monocomponent preparations, Ie , a component essentially free of other cellulolytic components The single component may be a recombinant component, i e , produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244) The host is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host) Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth

The cellulolytic proteins used in the methods of the present invention may be produced by fermentation of the above-noted microbial strains on a nutnent medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e g , Bennett, J W and LaSure, L (eds ), More Gene Manipulations in Fungi, Academic Press, CA, 1991) Suitable media are available from commercial suppliers or may be prepared according to published compositions (e g , in catalogues of the Amencan Type Culture Collection) Temperature ranges and other conditions suitable for growth and cellulolytic protein production are known in the art (see, e g , Bailey, J E , and Ollis, D F , Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986)

The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of a cellulolytic protein Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the cellulolytic protein to be expressed or isolated The resulting cellulolytic proteins produced by the methods descnbed above may be recovered from the fermentation medium and purified by conventional procedures as described herein

Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLUCLAST™ (available from Nσvozymes A/S) and NOVOZYM™ 188 (available from Novozymes A/S) Other commercially available preparations comprising cellulase that may be used include CELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ and SPEZYME™ CP (Genencor Int ), ROHAMENT™ 7069 W (Rδhm GmbH), and FIBREZYME® LDI, FIBREZYME® LBR, or VISCOSTAR® 150L (Dyadic International, Inc , Jupiter, FL, USA) The cellulase enzymes are added in amounts effective from about 0 001% to about 5 0 % wt of solids, more preferably from about 0 025% to about 4 0% wt of solids, and most preferably from about 0 005% to about 2 0% wt of solids The resulting cellulolytic proteins or beta-glucosidase proteins produced by the methods descnbed above may be recovered from the fermentation medium by conventional procedures including, but not limited to, centnfugation, filtration, spray- drying, evaporation, or precipitation The recovered protein may then be further purified by a variety of chromatographic procedures, e g , ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like

The activity of a cellulolytic protein can be determined using any method known in the art

Examples of cellulolytic preparations suitable for use in the present invention include, for example, CELLUCLAST™ (available from Novozymes A/S) and NOVOZYM™ 188 (available from Novozymes A/S) Other commercially available preparations comprising cellulase that may be used include CELLUZYME™ CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ and SPEZYME™ CP (Genencor Int ), and ROHAMENT™ 7069 W (Rδhm GmbH) The cellulase enzymes are added in amounts effective from about 0 001% to about 5 0 % wt of solids, more preferably from about 0 025% to about 4 0% wt of solids, and most preferably from about 0 005% to about 2 0% wt of solids

As mentioned above, the cellulolytic proteins used in the methods of the present invention may be monocomponent preparations, / e , a component essentially free of other cellulolytic components The single component may be a recombinant component, / e , produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244) Other examples of monocomponent cellulolytic proteins include, but are not limited to, those disclosed in JP-07203960-A and WO-9206209 The host is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host) Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth Examples of monocompoπeπt cellulolytic proteins useful in practicing the methods of the present invention include, but are not limited to, endoglucanase, cellobiohydrolase, and other enzymes useful in degrading cellulosic biomass

The term "endoglucanase" is already defined herein The term "cellobiohydrolase" is defined herein as a 1 ,4-beta-D-glucan cellobiohydrolase (E C 32 1 91), which catalyzes the hydrolysis of 1,4-beta-D-glucosιdιc linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-lιnked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain For purposes of the present invention, cellobiohydrolase activity can be determined according to the procedures described by Lever ef a/ , 1972, Anal Biochem 47 273-279 and by van Tilbeurgh er a/ , 1982, FEBS Letters 149 152-156, van Tilbeurgh and Claeyssens, 1985 FEBS Letters 187 283-288 In the present invention, the Lever ef al method was employed to assess hydrolysis of cellulose in corn stover

The polypeptides of the present invention are used in conjunction with cellulolytic proteins to degrade the cellulosic component of the biomass substrate, (see, for example, Bπgham ef al , 1995, in Handbook on Bioethanol (Charles E Wyman, editor), pp 119-141 , Taylor & Francis, Washington D C , Lee, 1997, Journal of Biotechnology 56 1-24)

The optimum amounts of a polypeptide having beta-glucosidase activity and of cellulolytic proteins depends on several factors including, but not limited to, the mixture of component cellulolytic proteins, the cellulosic substrate, the concentration of cellulosic substrate, the pretreatment(s) of the cellulosic substrate, temperature, time, pH, and inclusion of fermenting organism (e g , yeast for Simultaneous Saccharification and Fermentation) The term "cellulolytic proteins" is defined herein as those proteins or mixtures of proteins shown as being capable of hydrolyzing or converting or degrading cellulose under the conditions tested Their amounts are usually measured by a common assay such as BCA (bicinchoninic acid, P K Smith ef al , 1985, Anal Biochem 150 76), and the preferred amount added in proportion to the amount of biomass being hydrolyzed In a preferred aspect, the amount of polypeptide having beta-glucosidase activity per g of cellulosic material is about 001 to about 20 mg, preferably about 0025 to about 1 5 mg, more preferably about 005 to about 1 25 mg, more preferably about 0 075 to about 1 25 mg, more preferably about 0 1 to about 1 25 mg, even more preferably about 0 15 to about 1 25 mg, and most preferably about 025 to about 1 0 mg per g of cellulosic matenal

In another preferred aspect, the amount of cellulolytic proteins per g of cellulosic matenal is about 0 5 to about 50 mg, preferably about 0 5 to about 40 mg, more preferably about 05 to about 25 mg, more preferably about 075 to about 20 mg, more preferably about 0 75 to about 15 mg, even more preferably about 05 to about 10 mg, and most preferably about 25 to about 10 mg per g of cellulosic material

The methods of the present invention can be used to degrade or convert a cellulosic material, e g , lignocellulose, to many useful substances, e g , chemicals and fuels In addition to ethanol, some commodity and specialty chemicals that can be produced from cellulose include xylose, acetone, acetate, glycine, lysine, organic acids (e g , lactic acid), 1 ,3-propanedιol, butanediol, glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, and cis.cis-muconic acid (Lynd, L R , Wyman, C E , and Gerngross, T U , 1999, Biocommodity Engineering, Biotechnol Prog , 15 777-793, Philippidis, G P , 1996, Cellulose byconversion technology, in Handbook on Bioethanol Production and Utilization, Wyman, C E , ed , Taylor & Francis, Washington, DC, 179-212, and Ryu, D D Y , and Mandels, M , 1980, Cellulases biosynthesis and applications, Enz Microti Technol , 2 91-102) Potential coproduction benefits extend beyond the synthesis of multiple organic products from fermentable carbohydrate Lignin-rich residues remaining after biological processing can be converted to lignin-denved chemicals, or used for power production

Conventional methods used to process the cellulosic mateπal in accordance with the methods of the present invention are well understood to those skilled in the art The methods of the present invention may be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention

Such an apparatus may include a fed-batch stirred reactor, a batch-stirred reactor, a continuous flow stirred reactor with ultrafiltration, a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Sαentiarum Technology 25(1) 33-38, Gusakov, AV , and Sinitsyn, A P , 1985, Kinetics of the enzymatic hydrolysis of cellulose 1 A mathematical model for a batch reactor process, Enz Microb Technol 7 346-352), an attrition reactor (Ryu, S K , and Lee, J M , 1983, Biocon version of waste cellulose by using an attrition bioreactor, Biotechnol Bioeng 25 53-65), or a reactor with intensive stirnng induced by an electromagnetic field (Gusakov, A V , Sinitsyn, A P , Davydkin, I Y , Davydkin, V Y , Protas, O V , 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl Biochem Biotechnol 56 141-153) The conventional methods include, but are not limited to, sacchaπfication, fermentation, separate hydrolysis and fermentation (SHF), simultaneous sacchaπfication and fermentation (SSF), simultaneous saccharification and cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), and direct microbial conversion (DMC)

SHF uses separate process steps to first enzymatically hydrolyze cellulose to fermentable sugars and then, in a subsequent step, ferments sugars to ethanol In SSF, the enzymatic hydrolysis of cellulose and the fermentation of fermentable sugar to ethanol are combined in one step (Philippidis, G P , 1996, Cellulose byconversion technology, in Handbook on Bioethanol Production and Utilization, Wyman, C E , ed , Taylor & Francis, Washington, DC, 179-212) SSCF includes the cofermentation of multiple sugars (Sheehan, J , and Himmel, M , 1999, Enzymes, energy and the environment A strategic perspective on the U S Department of Energy's research and development activities for bioethanol, Biotechnot Prog 15 817-827) HHF includes two separate steps carried out in the same reactor but at different temperatures, i e , high temperature enzymatic sacchanfication followed by SSF at a lower temperature that the fermentation strain can tolerate DMC combines all three processes (cellulase production, cellulose hydrolysis, and fermentation) in one step (Lynd, L R , Weimer, P J , van ZyI, W H , and Pretonus, I S , 2002, Microbial cellulose utilization Fundamentals and biotechnology, Microbiol MoI Biol Reviews 66 506-577)

"Fermentation" or "fermentation process" refers to any fermentation process or any process compπsing a fermentation step A fermentation process includes, without limitation, fermentation processes used to produce fermentation products including alcohols (e g , arabimtol, butanol, ethanol, glycerol, methanol, 1,3-propanedιol, sorbitol, and xylitol), organic acids (e g , acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-dιketo-D-gluconιc acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaπc acid, 3-hydroxypropιonιc acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid), ketones (e g , acetone), ammo acids (e g , aspartic acid, glutamic acid, glycine, lysine, senne, and threonine), gases (e g , methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)) Fermentation processes also include fermentation processes used in the consumable alcohol industry (e g , beer and wine), dairy industry (e g , fermented dairy products), leather industry, and tobacco industry A fusion protein or a component thereof having cellulolytic activity or hemicellulolytic activity obtained according to the methods of the present invention, e g , a beta-glucosidase fusion protein or a component thereof, and host cells thereof, can be used in the production of monosacchaπdes, disaccharides, and polysaccharides as chemical or fermentation feedstocks from biomass for the production of ethanol, plastics, other products or intermediates In particular, the polypeptides and host cells may be used to increase the value of processing residues (dried distillers grain, spent grains from brewing, sugarcane bagasse, etc ) by partial or complete solubilization of cellulose or hemicellulose In boosting the processing of cellulosic material by the cellulolytic enzyme preparation to glucose, xylose, mannose, galactose, and arabinose, their polymers, or products derived from them as described below, the polypeptides may be in the form of a crude fermentation broth with or without the cells or in the form of a semi-purified or purified enzyme preparation The polypeptide may be a monocomponent preparation, a multicomponent protein preparation, or a combination of multicomponent and monocomponent protein preparations Alternatively, a host cell may be used as a source of such a polypeptide in a fermentation process with the biomass The host cell may also contain native or heterologous genes that encode cellulolytic protein as well as other enzymes useful in the processing of biomass

The present invention further relates to methods for producing an organic substance, comprising (a) saccharifying a cellulosic matenal with an effective amount of a cellulolytic enzyme composition i in the presence of an effective amount of a fusion protein or a component thereof having cellulolytic activity or hemicellulolytic activity obtained according to the instant methods, (b) fermenting the saccharified cellulosic matenal of step (a) with one or more fermenting microorganisms, and (c) recovering the organic substance from the fermentation As indicated earlier, for purposes of illustration, a polypeptide having beta-glucosidase activity obtained according to the methods of the present invention, e g , a beta-glucosidase fusion protein or a component thereof is used for illustrative purposes The polypeptide having beta-glucosidase activity may be in the form of a crude fermentation broth with or without the cells or in the form of a semi- punfied or purified enzyme preparation The beta-glucosidase protein may be a monocomponent preparation, a multicomponent protein preparation, or a combination of multicomponent and monocomponent protein preparations The substance can be any substance derived from the fermentation In a preferred aspect, the substance is an alcohol It will be understood that the term "alcohol' encompasses a substance that contains one or more hydroxy I moieties In a more preferred aspect, the alcohol is arabinitol In another more preferred aspect, the alcohol is butanol In another more preferred aspect, the alcohol is ethanol In another more preferred aspect, the alcohol is glycerol In another more preferred aspect, the alcohol is methanol In another more preferred aspect, the alcohol is 1 ,3-proρanedιol In another more preferred aspect, the alcohol is sorbitol In another more preferred aspect, the alcohol is xylitol See, for example, Gong, C S , Cao, N J , Du, J , and Tsao, G T , 1999, Ethanol production from renewable resources, in Advances in Biochemical Engmeeπng/Botechnology, Scheper, T , ed , Spπnger-Verlag Berlin Heidelberg, Germany, 65 207-241, Silveira, M M , and Jonas, R , 2002, The biotechnological production of sorbitol, Appl Microbiol Biotechnol 59 400-408, Nigam, P , and Singh, D , 1995, Processes for fermentative production of xylitol - a sugar substitute, Process Biochemistry 30 (2) 117-124, Ezeji, T C , Qureshi, N and Blaschek, H P , 2003, Production of acetone, butanol and ethanol by Clostridium beijennckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6) 595-603

In another preferred aspect, the substance is an organic acid In another more preferred aspect, the organic acid is acetic acid In another more preferred aspect, the organic acid is acetonic acid In another more preferred aspect, the organic acid is adipic acid In another more preferred aspect, the organic acid is ascorbic acid In another more preferred aspect, the organic acid is citric acid In another more preferred aspect, the organic acid is 2,5-dιketo-D-gluconιc acid In another more preferred aspect, the organic acid is formic acid In another more preferred aspect, the organic acid is fumaπc acid In another more preferred aspect, the organic acid is glucaπc acid In another more preferred aspect, the organic acid is gluconic acid In another more preferred aspect, the organic acid is glucuronic acid In another more preferred aspect, the organic acid is glutaric acid In another preferred aspect, the organic acid is 3- hydroxypropionic acid In another more preferred aspect, the organic acid is itaconic acid In another more preferred aspect, the organic acid is lactic acid In another more preferred aspect, the organic acid is malic acid In another more preferred aspect, the organic acid is malonic acid In another more preferred aspect, the organic acid is oxalic acid In another more preferred aspect, the organic acid is propionic acid In another more preferred aspect, the organic acid is succinic acid In another more preferred aspect, the organic acid is xylonic acid See, for example, Chen, R , and Lee, Y Y , 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl Biochem Biotechnol 63-65 435-448

In another preferred aspect, the substance is a ketone It will be understood that the term "ketone" encompasses a substance that contains one or more ketone moieties In another more preferred aspect, the ketone is acetone See, for example, Qureshi and Blaschek, 2003, supra In another preferred aspect, the substance is an ammo acid In another more preferred aspect, the organic acid is aspartic acid In another more preferred aspect, the amino acid is glutamic acid In another more preferred aspect, the amino acid is glycine In another more preferred aspect, the amino acid is lysine In another more preferred aspect, the amino acid is senne In another more preferred aspect, the amino acid is threonine See, for example, Richard, A , and Margaπtis, A , 2004, Empirical modeling of batch fermentation kinetics for poly(glutamιc acid) production and other microbial biopolymers, Biotechnology and Bioengineenng 87 (4) 501-515 In another preferred aspect, the substance is a gas In another more preferred aspect, the gas is methane In another more preferred aspect, the gas is H₂ In another more preferred aspect, the gas is CO₂ In another more preferred aspect, the gas is CO See, for example, Kataoka, N , A Miya, and K Kiπyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bactena, Water Science and Technology 36 (6-7) 41-47, and Gunaseelan V N in Biomass and Bioenergy, VoI 13 (1-2), pp 83-114, 1997, Anaerobic digestion of biomass for methane production A review

Production of a substance from cellulosic material typically requires four major steps These four steps are pretreatment, enzymatic hydrolysis, fermentation, and recovery Exemplified below is a process for producing ethanol, but it will be understood that similar processes can be used to produce other substances, for example, the substances described above

Prelreatment In the pretreatment or pre-hydrolysis step, the cellulosic material is heated to break down the lignin and carbohydrate structure, solubilize most of the hemicellulose, and make the cellulose fraction accessible to cellulolytic enzymes The heating is performed either directly with steam or in slurry where a catalyst may also be added to the material to speed up the reactions Catalysts include strong acids, such as sulfuric acid and SO₂, or alkali, such as sodium hydroxide The purpose of the pre- treatment stage is to facilitate the penetration of the enzymes and microorganisms Cellulosic biomass may also be subject to a hydrothermal steam explosion pretreatment (See U S Patent Application No 20020164730) However, it is understood that in practicing the methods of the present invention, any pretreatment can be used employing thermal, chemical, and/or mechanical pretreatment Sacchaπfication In the enzymatic hydrolysis step, also known as sacchaπfication, enzymes as descnbed herein are added to the pretreated material to convert the cellulose fraction to glucose and/or other sugars The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions A saccharification step may last up to 200 hours Saccharification may be carried out at temperatures from about 30°C to about 65°C, in particular around 50°C, and at a pH in the range between about 4 to about 5, especially around pH 4 5 To produce glucose that can be metabolized by yeast, the hydrolysis is typically performed in the presence of a beta-glucosidase

Fermentation In the fermentation step, sugars, released from the cellulosic mateπal as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to ethanol by a fermenting organism, such as yeast The fermentation can also be earned out simultaneously with the enzymatic hydrolysis in the same vessel, again under controlled pH, temperature, and mixing conditions When sacchaπfication and fermentation are performed simultaneously in the same vessel, the process is generally termed simultaneous sacchaπfication and fermentation or SSF

Any suitable cellulosic material or raw material may be used in the fermentation step in practicing the present invention The material is generally selected based on the desired fermentation product, / e , the substance to be obtained from the fermentation, and the process employed, as is well known in the art Examples of substrates suitable for use in the methods of present invention, include cellulose-containing materials, such as wood or plant residues or low molecular sugars DP1-3 obtained from processed cellulosic material that can be metabolized by the fermenting microorganism, and which may be supplied by direct addition to the fermentation medium

The term "fermentation medium" will be understood to refer to a medium before the fermenting mιcroorganιsm(s) ιs(are) added, such as, a medium resulting from a sacchaπfication process, as well as a medium used in a simultaneous sacchaπfication and fermentation process (SSF)

"Fermenting microorganism^* refers to any microorganism suitable for use in a desired fermentation process Suitable fermenting microorganisms are able to ferment, i e , convert, sugars, such as glucose, xylose, arabinose, mannose, galactose, or oligosaccharides directly or indirectly into the desired fermentation product Examples of fermenting microorganisms include fungal organisms, such as yeast Preferred yeast includes strains of the Saccharomyces spp , and in particular, Saccharomyces cerevisiae Commercially available yeast include, e g , RED STAR®/Lesaffre Ethanol Red (available from RED STAR®/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a division of Burns Philp Food Inc , USA), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL (available from DSM Specialties)

In a preferred aspect, the yeast is a Saccharomyces spp In a more preferred aspect, the yeast is Saccharomyces cerevisiae In another more preferred aspect, the yeast is Saccharomyces distattcus In another more preferred aspect, the yeast is Saccharomyces uvarum In another preferred aspect, the yeast is a Kluyveromyces In another more preferred aspect, the yeast is Kluyveromyces marxianus In another more preferred aspect, the yeast is Kluyveromyces fragilis In another preferred aspect, the yeast is a Candida In another more preferred aspect, the yeast is Candida pseudotropicalis In another more preferred aspect, the yeast is Candida brassicae In another preferred aspect, the yeast is a Clavispora In another more preferred aspect, the yeast is Clavispora lusitaniae In another more preferred aspect, the yeast is Clavispora opuntiae In another preferred aspect, the yeast is a Pachysolen In another more preferred aspect, the yeast is Pachysolen tannophilus In another preferred aspect, the yeast is a Bretannomyces In another more preferred aspect, the yeast is Bretannomyces dausenn (Philippidis, G P , 1996, Cellulose byconversion technology, in Handbook on Bioethanol Production and Utilization, Wyman, C E , ed , Taylor & 5 Francis, Washington, DC, 179-212)

Bacteria that can efficiently ferment glucose to ethanol include, for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra)

It is well known in the art that the organisms described above can also be used to produce other substances, as described herein

10 The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z , Ho

N W Y , 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl Biochem Biotechnol 39-40 135-147, Ho, N W Y , Chen, Z, Brainard, A P , 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl Environ Microbiol 64

I₅ 1852-1859), or in bactena such as Escheπchia coli (Beall, D S , Ohta, K , Ingram, L O , 1991 , Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coil, Biotech Bioeng 38 296-303), Klebsiella oxytoca (Ingram, L O , Gomes, P F , Lai, X , Momruzzaman, M , Wood, B E , Yomano, L P , York, S W , 1998, Metabolic engineeπng of bacteria for ethanol production, Biotechnol Bioeng

20 58 204-214), and Zymomonas mobihs (Zhang, M , Eddy, C , Deanda, K , Finkelstein, M , and Picataggio, S , 1995, Metabolic engineenng of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267 240-243, Deanda, K , Zhang, M , Eddy, C , and Picataggio, S , 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl Environ Microbiol

25 62 4465-4470) has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (cofermentation)

Yeast or another microorganism typically is added to the degraded cellulose or hydrolysate and the fermentation is performed for about 24 to about 96 hours, such as about 35 to about 60 hours The temperature is typically between about 26⁰C to about

30 40⁰C, in particular at about 32°C, and at about pH 3 to about pH 6, in particular around pH 4-5

In a preferred aspect, yeast or another microorganism is applied to the degraded cellulose or hydrolysate and the fermentation is performed for about 24 to about 96 hours, such as typically 35-60 hours In a preferred aspect, the temperature is generally

35 between about 26 to about 40⁰C, in particular about 32⁰C, and the pH is generally from about pH 3 to about pH 6, preferably around pH 4-5 Yeast or another microorganism is preferably applied in amounts of approximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰, especially approximately 5x10⁷ viable cell count per ml of fermentation broth During an ethanol producing phase the yeast cell count should preferably be in the range from approximately 10⁷ to 10¹⁰, especially around approximately 2 x 10⁸ Further guidance in respect of using yeast for fermentation can be found in, e g , "The Alcohol Textbook" (Editors K Jacques, T P Lyons and D R Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference

The most widely used process in the art is the simultaneous saccharification and fermentation (SSF) process where there is no holding stage for the saccharification, meaning that yeast and enzyme are added together

For ethanol production, following the fermentation the mash is distilled to extract the ethanol The ethanol obtained according to the process of the invention may be used as, e g , fuel ethanol, drinking ethanol, / e , potable neutral spirits, or industrial ethanol A fermentation stimulator may be used in combination with any of the enzymatic processes descπbed herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield A "fermentation stimulator" refers to stimulators for growth of the fermenting microorganisms, in particular, yeast Preferred fermentation stimulators for growth include vitamins and minerals Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyndoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E See, for example, Alfenore ef a/ , Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy duπng fed-batch process, Spπnger-Verlag (2002), which is hereby incorporated by reference Examples of minerals include minerals and mineral salts that can supply nutnents comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu

Recovery The alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation Ethanol with a purity of up to about 96 vol % can be obtained, which can be used as, for example, fuel ethanol, dnnking ethanol, / e , potable neutral spirits or industrial ethanol

For other substances, any method known in the art can be used including, but not limited to, chromatography (e g , ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e g , preparative isoelectric focusing), differential solubility (e g , ammonium sulfate precipitation), SDS- PAGE, distillation, or extraction

In the methods of the present invention, besides beta-glucosidase, the cellulolytic enzyme preparation and cellulolytic enhancing polypeptιde(s) may be supplemented by one or more additional enzyme activities to improve the degradation of the cellulosic mateπal Preferred additional enzymes are hemicellulases, esterases (e g , lipases, phosphohpases, and/or cutinases), proteases, laccases, peroxidases, or mixtures thereof

In the methods of the present invention, the additional enzyme(s) may be added prior to or during fermentation, including duπng or after the propagation of the fermenting mιcroorganιsm(s)

The enzymes referenced herein may be derived or obtained from any suitable origin, including, bacterial, fungal, yeast or mammalian origin The term "obtained^* means herein that the enzyme may have been isolated from an organism that naturally produces the enzyme as a native enzyme The term "obtained" also means herein that the enzyme may have been produced recombinant^ in a host organism, wherein the recombinant^ produced enzyme is either native or foreign to the host organism or has a modified ammo acid sequence, e g , having one or more amino acids that are deleted, inserted and/or substituted, / e , a recombinant^ produced enzyme that is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are vaπants obtained recombinant^, such as by site-directed mutagenesis or shuffling

The enzymes may also be purified The term "purified" as used herein covers enzymes free from other components from the organism from which it is denved The term "purified" also covers enzymes free from components from the native organism from which it is obtained The enzymes may be puπfied, with only minor amounts of other proteins being present The expression "other proteins" relate in particular to other enzymes The term "purified" as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of oπgin of the enzyme of the invention The enzyme may be "substantially pure polypeptide," that is, free from other components from the organism in which it is produced, that is, for example, a host organism for recombinants produced enzymes

The enzymes used in the present invention may be in any form suitable for use in the processes described herein, such as, for example, a crude fermentation broth with or without cells, a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme Granulates may be produced, e g , as disclosed in U S Patent Nos 4,106,991 and 4,661 ,452, and may optionally be coated by process known in the art Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established process Protected enzymes may be prepared according to the process disclosed in EP 238,216

Detergent Compositions

5 The methods of the present invention are particularly useful for improving the secretion of polypeptides in commercially important quantities for use in detergent compositions Such polypeptides include, but are not limited to proteases, cellulolytic enzymes, amylases, and peroxidases, or any other enzyme or biological protein useful to the detergent industry

10 The detergent composition of the present invention may be, for example, formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabπcs and a rinse added fabnc softener composition, or formulated as a detergent composition for use in general household hard surface cleaning operations, or formulated for hand or machine

I₅ dishwashing operations

In a specific aspect, the present invention provides a detergent additive comprising a polypeptide having biological activity (e g , fusion protein, a component thereof, or combinations thereof) obtained according to the present invention The detergent additive as well as the detergent composition may compπse one or more other enzymes such as a

20 protease, lipase, cutinase, an amylase, carbohydrase cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e g , a laccase, and/or peroxidase

In general the properties of the enzymatic components should be compatible with the selected detergent, (/ e , pH optimum, compatibility with other enzymatic and non- enzymatic ingredients, etc), and the enzymatic components should be present in effective

25 amounts

Proteases Suitable proteases include those of animal, vegetable or microbial origin Microbial ongin is preferred Chemically modified or protein engineered mutants are included The protease may be a seπne protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease Examples of alkaline proteases

30 are subtihsins, especially those denved from Bacillus, e g , subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) Examples of trypsin-like proteases are trypsin (e g , of porcine or bovine origin) and the Fusaπum protease described in WO 89/06270 and WO 94/25583

Examples of useful proteases are the vaπants described in WO 92/19729, WO

35 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274 Preferred commercially available protease enzymes include ALCALASE™, SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, AND KANNASE™ (NOVOZYMES A/S), MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™, FN2™, and FN3™ (Genencor International lnc ) Lipases Suitable lipases include those of bacteπal or fungal origin Chemically modified or protein engineered mutants are included Examples of useful lipases include lipases from Humicola (synonym Thermomyces) e g , from H lanuginosa (T lanugmosus) as described in EP 258 068 and EP 305 216 or from H insolens as described in WO 96/13580, a Pseudomonas lipase, e g , from P alcaligenes or P pseudoalcaligenes (EP 218 272), P cepacia (EP 331 376), P stutzeπ (GB 1 ,372,034), P fluorescens, Pseudomonas sp strain SD 705 (WO 95/06720 and WO 96/27002), P wisconsinensis (WO 96/12012), a Bacillus lipase, e g , from B subtihs (Dartois et al , 1993, Biochemica et Biophysica Acta, 1131 , 253-360), B stearothermophilus (JP 64/744992) or B pumilus (WO 91/16422) Other examples are lipase variants such as those described in WO 92/05249,

WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783 WO 95/22615, WO 97/04079 and WO 97/07202

Preferred commercially available lipases include LIPOLASE™, LIPEX™, and Lipolase ULTRA™ (Novozymes A/S) Amylases Suitable amylases (α and/or β) include those of bacterial or fungal origin Chemically modified or protein engineered mutants are included Amylases include, for example α-amylases obtained from Bacillus, e g , a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International lnc )

Cellulases Suitable cellulases include those of bacteπal or fungal origin Chemically modified or protein engineered mutants are included Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusanum, Thielavia, Acremonium, or Tnchoderma e g , the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusanum oxysporum disclosed in U S Patent No 4,435,307, U S Patent No 5,648,263, U S Patent No 5,691 ,178, U S Patent No 5,776,757 and WO 89/09259 Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940 Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U S Patent No 5,457,046, U S Patent No 5,686,593, U S Patent No 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299

Commercially available cellulases include CELLUCLAST®, CELLUZYME™, and CAREZYME™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International lnc ), and KAC-500(B)™ (Kao Corporation) Peroxidases/Oxidases Suitable peroxidases/oxidases include those of plant, bactenal or fungal origin Chemically modified or protein engineered mutants are included Examples of useful peroxidases include peroxidases from Copnnus, e g , from C anereus, and vanants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257 Commercially available peroxidases include GUARDZYME™ (Novozymes A/S)

The enzymatic component(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive compπsing all of these enzymes A detergent additive of the present invention, i e , a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries

Non-dusting granulates may be produced, e g , as disclosed in U S Patent Nos 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art Examples of waxy coating matenals are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000, ethoxylated nonylphenols having from 16 to 50 ethylene oxide units, ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units, fatty alcohols fatty acids, and mono- and dι- and tnglyceπdes of fatty acids Examples of film-forming coating matenals suitable for application by fluid bed techniques are given in GB 1483591 Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or bone acid according to established methods Protected enzymes may be prepared according to the method disclosed in EP 238,216

The detergent composition of the present invention may be in any convenient form, e g , a bar, a tablet, a powder, a granule, a paste or a liquid A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous

The detergent composition comprises one or more surfactants, which may be non- ionic including semi-polar and/or anionic and/or cation ic and/or zwittenoπic The surfactants are typically present at a level of from 0 1 % to 60% by weight

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, 5 alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha- sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap

When included therein the detergent will usually contain from about 0 2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,

10 fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides")

The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nrtπlotπacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or

I₅ alkenylsuccinic acid, soluble silicates or layered silicates (e g , SKS-6 from Hoechst)

The detergent may compnse one or more polymers EExamples are carboxymethylcellulose, polyvinylpyrrolidone), polyethylene glycol), poly(vιnyl alcohol), poly(vιnylpyπdιne-N-oxιde), poly(vιnylιmιdazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acid copolymers

20 The detergent may contain a bleaching system that may compnse a H₂O₂ source such as perborate or percarbonate that may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate Alternatively, the bleaching system may compnse peroxyacids of, for example, the amide, imide, or sulfone type

25 The enzymatic component(s) of the detergent composition of the present invention may be stabilized using conventional stabilizing agents, e g , a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, bone acid, or a boric acid denvative, e g , an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO

30 92/19709 and WO 92/19708

The detergent may also contain other conventional detergent ingredients such as fabnc conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical bπghteners, hydrotropes, tarnish inhibitors, or perfumes

35 In the detergent compositions any enzymatic component, in particular the polypeptides having biological activity of the present invention, may be added in an amount corresponding to 001-100 mg of enzyme protein per liter of wash liquor, preferably 005-5 mg of enzyme protein per liter of wash liquor, in particular 0 1-1 mg of enzyme protein per liter of wash liquor

The polypeptides having biological activity of the present invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202, which is hereby incorporated as reference

Other Uses

Polypeptides having biological activity (e g , fusion protein, a component thereof or combinations thereof) obtained according to the present invention o may also be used in combination with other glycohydrolases and related enzymes, as descnbed herein, in the treatment of textiles as biopolishing agents and for reducing of fuzz, pilling, texture modification, and stonewashing (N K Lange, in P Suominen, T Reimkainen (Eds ), Trichoderma reesei Cellulases and Other Hydrolases, Foundation for Biotechnical and Industπal Fermentation Research, Helsinki, 1993, pp 263-272) In addition, the described polypeptides may also be used in combination with other glycohydrolases and related enzymes, as described herein, in wood processing for biopulping or debarking, paper manufacturing for fiber modification, bleaching, and reduction of refining energy costs, Whitewater treatment, important to wastewater recycling, lignocellulosic fiber recycling such as deinking and secondary fiber processing, and wood residue utilization (S D Mansfield and A R Esteghlalian in S D, Mansfield and J N Saddler (Eds ), Applications of Enzymes to ϋgnocellulosics, ACS Symposium Series 855, Washington, D C , 2003 pp 2-29)

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention

Examples

Chemicals used as buffers and substrates were commercial products of at least reagent grade

DNA Sequencing

DNA sequencing was performed using an Applied Biosystems Model 3130X

Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using dye terminator chemistry (Giesecke et a/ , 19Θ2, Journal of Virol Methods 38 47-60) Sequences were

5 assembled using phred/phrap/consed (University of Washington, Seattle, WA, USA) with sequence specific primers

Strain

Tnchoderma reesei RutC30 (ATCC 56765, Montenecourt and Eveleigh, 1979

10 Adv Chem Ser 181 289-301) was derived from Tnchoderma reesei Qm6A (ATCC

13631 , Mandels and Reese, 1957, J Baderiol 73 269-278) Tnchoderma reesei

RutC30 and Aspergillus oryzae Jal355 strain (WO 02/062973) were used for expression of the beta-glucosidase fusion protein

I₅ Media and Solutions

YP medium was composed per liter of 10 g of yeast extract and 20 g of bacto tryptone

Cellulase-inducing medium was composed per liter of 20 g of cellulose, 10 g of corn steep solids, 1 45 g of (NH₄)_JSO₄, 2 08 g of KH₂PO₄, 028 g of CaCI₂, 042 g of 20 MgSO₄7H₂O, and 042 ml of trace metals solution

Trace metals solution was composed per liter of 216 g of FeCI₃6H₂O, 58 g of ZnSO₄7H₂O, 27 g of MnSO₄ H₂O, 10 g of CuSO₄5H₂O, 24 g of H₃BO₃, and 336 g of citric acid

STC was composed of 1 M sorbitol, 10 mM CaCI₂, and 10 mM Tns-HCI, pH 75 25 COVE plates were composed per liter of 342 g of sucrose, 10 ml of COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 1 5 M CsCI, and 25 g of Noble agar

COVE salts solution was composed per liter of 26 g of KCI, 26 g of MgSO₄, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution

COVE trace metals solution was composed per liter of 0 04 g of Na₂B₄O₇ 10H₂O, 30 04 g of CuSO₄5H₂O, 1 2 g of FeSO₄7H₂O, 07 g of MnSO₄ H₂O, 0 8 g of Na₂MoO₂ H₂O, and 10 g Of ZnSO₄7H₂O

COVE2 plates were composed per liter of 30 g of sucrose, 20 ml of COVE salts solution, 25 g of Noble agar, and 10 ml of 1 M acetamide

PDA plates were composed per liter of 39 grams of potato dextrose agar 35 LB medium was composed per liter of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloπde

2X YT plates were composed per liter of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, and 15 g of Bacto Agar

MDU2BP medium was composed per liter of 45 g of maltose, 1 g of MgSO₄ 7H₂O, 1 g of NaCI, 2 g of K₂HSO₄, 12 g of KH₂PO₄, 2 g of urea, and 500 μl of AMG trace metals solution, the pH was adjusted to 5 O and then filter sterilized with a 5 O 22 μm filtering unit

AMG trace metals solution was composed per liter of 14 3 g of ZnSO₄ 7H₂O, 25 g of CuSO₄5H₂O, O 5 g of NiCI₂ 6H₂O, 138 g of FeSO₄ 7H₂O, 85 g of MnSO₄7H₂O, and 3 g of citric acid

Minimal medium plates were composed per liter of 6 g of NaNO₃, O 52 of KCI, 10 1 52 g of KH₂PO₄, 1 ml of COVE trace metals solution, 20 g of Noble agar, 20 ml of 50% glucose, 25 ml of 20% MgSO₄7H₂O, and 20 ml of biotin stock solution

Biotin stock solution was composed per liter of O 2 g of biotin

SOC medium was composed of 2% tryptone, O 5% yeast extract, 10 mM NaCI, 2 5 mM KCI, 10 mM MgCI₂, and 10 mM MgSO₄, followed by addition of filter-sterilized I₅ glucose to 20 mM after autoclaving

Example 1: Construction of pMJ04 expression vector

Expression vector pMJ04 was constructed by PCR amplifying the Trichoderma reesei cellobiohydrolase 1 gene (cbM, CEL7A) terminator from Trichoderma reesei

20 RutC30 genomic DNA using primers 993429 (antisense) and 993428 (sense) shown below The antisense primer was engineered to have a Pac I site at the 5'-end and a Spe I site at the 3'-end of the sense pnmer Primer 993429 (antisense) S'-AACGTTAATTAAGGAATCGTTTTGTGTTT-S' (SEQ ID NO 33)

2₅ Primer 993428 (sense) δ'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-S' (SEQ ID NO 34)

Trichoderma reesei RutC30 genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN lnc , Valencia, CA, USA)

The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction

30 Buffer (New England Biolabs, Beverly, MA USA), 03 mM dNTPs 100 ng of Trichoderma reesei RutC30 genomic DNA, O 3 μM pnmer 993429, O 3 μM primer 993428, and 2 units of Vent DNA polymerase (New England Biolabs, Beverly, MA, USA) The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, lnc , Westbury, NY, USA) programmed for 5 cycles each for 30

3₅ seconds at 94°C, 30 seconds at 5O⁰C, and 60 seconds at 72⁰C, followed by 25 cycles each for 30 seconds at 94°C, 30 seconds at 65°C, and 120 seconds at 72°C (5 minute final extension) The reaction products were isolated on a 1 0% agarose gel using 40 mM Tπs base-20 mM sodium acetate- 1 mM disodium EDTA (TAE) buffer where a 229 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit (QIAGEN lnc , Valencia, CA, USA) according to the manufacturer's instructions The resulting PCR fragment was digested with Pac I and Spe I and hgated into pAILoi (WO 05/067531) digested with the same restriction enzymes using a Rapid Ligation Kit (Roche, Indianapolis, IN, USA), to generate pMJ04 (Figure 1)

Example 2: Construction of pCaHj568 Plasmid pCaHj568 was constructed from pCaHj170 (U S Patent No 5,763,254) and pMT2188 Plasmid pCaHj170 comprises the Humicola insolens endoglucanase V (CEL45A) full-length coding region (SEQ ID NO 1, which encodes the amino acid sequence of SEQ ID NO 2) Construction of pMT2188 was initiated by PCR amplifying the pUC19 origin of replication from pCaHj483 (WO 98/00529) using primers 142779 and 142780 shown below Primer 142780 introduces a Bbu I site in the PCR fragment 142779

S'-TTGAATTGAAAATAGATTGATTTAAAACTTC-S' (SEQ ID NO 35) 142780 5'-TTGCATGCGTAATCATGGTCATAGC-S' (SEQ ID NO 36) An EXPAND® PCR System (Roche Molecular Biochemicals, Basel, Switzerland) was used following the manufacturer's instructions for this amplification PCR products were separated on an agarose gel and an 1160 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit (Genomed, Wielandstr, Germany) The URA3 gene was amplified from the general Saccharomyces cerevisiae cloning vector pYES2 (Invitrogen, Carlsbad, CA, USA) using primers 140288 and 142778 shown below using an EXPAND® PCR System Primer 140288 introduced an Eco Rl site into the PCR fragment

S'-TTGAATTCATGGGTAATAACTGATAT-S' (SEQ ID NO 37) 142778 5'-AAATCAATCTATTTTCAATTCAATTCATCATT-S' (SEQ ID NO 38)

PCR products were separated on an agarose gel and an 1126 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit

The two PCR fragments were fused by mixing and amplifed using primers 142780 and 140288 shown above by the overlap splicing method (Morton et al , 1989, Gene 77 61-68) PCR products were separated on an agarose gel and a 2263 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit

The resulting fragment was digested with Eco Rl and Bbu I and hgated using standard protocols to the largest fragment of pCaHj483 digested with the same restπction enzymes The ligation mixture was transformed into pyrF-negative E coll strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa,

1970, J MoI Biol 45 154 Transformants were selected on solid M9 medium (Sambrook et a/ , 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold

Spring Harbor Laboratory Press) supplemented per liter with 1 g of casamino acids, 500 μg of thiamine, and 10 mg of kanamycin A plasmid from one transformant was isolated and designated pCaMj527 (Figure 2)

The NA2-tpι promoter present on pCaMj527 was subjected to site-directed mutagenesis by a simple PCR approach using an EXPAND® PCR System according to the manufacturer's instructions Nucleotides 134-144 were converted from

GTACTAAAACC (SEQ ID NO 39) to CCGTTAAATTT (SEQ ID NO 40) using mutagenic primer 141223 shown below

Primer 141223 δ'-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC-S' (SEQ ID

NO 41)

Nucleotides 423-436 were converted from ATGCAATTTAAACT (SEQ ID NO 42) to

CGGCAATTTAACGG (SEQ ID NO 43) using mutagenic primer 141222 shown below

Primer 141222 δ'-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-S' (SEQ ID NO

44)

The resulting plasmid was designated pMT2188 (Figure 3)

The Humicola msolens endoglucanase V coding region was transferred from ρCaHj170 as a Bam Ml-Sa/ 1 fragment into pMT2188 digested with Bam HI and Xho I to generate pCaHj568 (Figure 4) Plasmid pCaHj568 comprises a mutated NA2-tpι promoter operably linked to the Humicola msolens endoglucanase V full-length coding sequence

Example 3: Construction of pMJOS Plasmid pMJ05 was constructed by PCR amplifying the 915 bp Humicola msolens endoglucanase V full-length coding region from pCaHj568 using primers

HiEGV-F and HiEGV-R shown below

HiEGV-F (sense) δ'-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-S' (SEQ ID NO 45) HiEGV-R (antisense)

S'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-S' (SEQ ID NO 46)

The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction Buffer (New England Biolabs, Beverly, MA, USA), 03 πM dNTPs, 10 ng/μl of pCaHj568, 0 3 μM HiEGV-F pπmer, 0 3 μM HiEGV-R primer, and 2 units of Vent DNA polymerase (New England Biolabs, Beverly, MA, USA) The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C, 30 seconds at 5O⁰C, and 60 seconds at 72⁰C, followed by 25 cycles each for 30 seconds at 94°C, 30 seconds at 65°C, and 120 seconds at 72°C (5 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 937 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions The 937 bp purified fragment was used as template DNA for subsequent amplifications using the following primers HiEGV-R (antisense)

S'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-S' (SEQ ID NO 47) HiEGV-F-overlap (sense) S'-ACCGCGGACTGCGCA TCATGCGTTCCTCCCCCCTCC-S' (SEQ ID NO 48)

Primer sequences in italics are homologous to 17 bp of the Tnchoderma reesei cellobiohydrolase I gene (cbM) promoter and underlined primer sequences are homologous to 29 bp of the Hum/cola insolens endoglucanase V coding region A 36 bp overlap between the promoter and the coding sequence allowed precise fusion of a 994 bp fragment comprising the Tnchoderma reesei cbM promoter to the 918 bp fragment compπsing the Humicola insolens endoglucanase V coding region

The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction Buffer, 0 3 mM dNTPs, 1 μl of the purified 937 bp PCR fragment, 0 3 μM HiEGV-F- overlap primer, 03 μM HiEGV-R primer, and 2 units of Vent DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C, 30 seconds at 50°C, and 60 seconds at 72^CC, followed by 25 cycles each for 30 seconds at 94⁰C, 30 seconds at 65°C, and 120 seconds at 72°C (5 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 945 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

A separate PCR was performed to amplify the Tnchoderma reesei cbM promoter sequence extending from 994 bp upstream of the ATG start codon of the gene from Tnchoderma reesei RutC30 genomic DNA using the pπmers shown below (the sense pπmer was engineered to have a Sa/ 1 restriction site at the 5'-end) Tnchoderma reesei RutC30 genomic DNA was isolated using a DNEASY® Plant Maxi Kit TrCBHIpro-F (sense) 5^■-AAACGTCGACCGAATGTAGGATTGTTATC-3^■ (SEQ ID NO 49) TrCBHIpro-R (antiseπse) δ'-GATGCGCAGTCCGCGGT-S' (SEQ ID NO 50)

The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction 5 Buffer, 0 3 mM dNTPs, 100 ng/μl Tπchoderma reesei RutC30 genomic DNA, 03 μM TrCBHIpro-F primer, 0 3 μM TrCBHIpro-R primer, and 2 units of Vent DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94⁰C, 30 seconds at 55⁰C, and 120 seconds at 72°C (5 minute final extension) The reaction products were isolated on a

10 1 0% agarose gel using TAE buffer where a 998 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

The purified 998 bp PCR fragment was used as template DNA for subsequent amplifications using the pnmers shown below

I₅ TrCBHIpro-F δ'-AAACGTCGACCGAATGTAGGATTGTTATC-S' (SEQ ID NO 51) TrCBHIpro-R-overlap S'-GGAGGGGGGAGGAACGCATGATGCGCΛGTCCGCGGT-S' (SEQ ID NO 52)

Sequences in italics are homologous to 17 bp of the Trichoderma reesei cbM

20 promoter and underlined sequences are homologous to 29 bp of the Humicola insolens endoglucanase V coding region A 36 bp overlap between the promoter and the coding sequence allowed precise fusion of the 994 bp fragment comprising the Trichoderma reesei cbM promoter to the 918 bp fragment comprising the Humicola insolens endoglucanase V full-length coding region

25 The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction

Buffer, 0 3 mM dNTPs, 1 μl of the puπfied 998 bp PCR fragment, 0 3 μM TrCBHI pro-F primer, 03 μM TrCBHI pro-R-overlap pπmer, and 2 units of Vent DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C, 30 seconds at 50⁰C, and 60 seconds at 72°C,

30 followed by 25 cycles each for 30 seconds at 94⁰C, 30 seconds at 65°C, and 120 seconds at 72°C (5 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 1017 bp product band was excised from the gel and puπfied using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

3₅ The 1017 bp Tnchoderma reesei cbh1 promoter PCR fragment and the 945 bp

Humicola insolens endoglucanase V PCR fragment were used as template DNA for subsequent amplification using the following pnmers to precisely fuse the 994 bp cbM promoter to the 918 bp eπdoglucaπase V full-length coding region using overlapping PCR

TrCBHIpro-F

5^•-AAACGTCGACCGAATGTAGGATTGTTATC-3^• (SEQ ID NO 53) HiEGV-R δ'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-S' (SEQ ID NO 54)

The amplification reactions (50 μl) were composed of 1X ThermoPol Reaction Buffer, 0 3 mM dNTPs, 03 μM TrCBH1pro-F primer, 03 μM HiEGV-R primer, and 2 units of Vent DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C, 30 seconds at 50⁰C, and 60 seconds at 72°C, followed by 25 cycles each for 30 seconds at 94°C, 30 seconds at 65⁰C, and 120 seconds at 72°C (5 minute fi nal extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 1926 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

The resulting 1926 bp fragment was cloned into a pCR®-Blunt-ll-TOPO® vector (Invitrogen, Carlsbad, CA, USA) using a ZEROBLUNT® TOPO® PCR Cloning Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol The resulting plasmid was digested with Not I and Sal I and the 1926 bp fragment was gel purified using a QIAQUICK® Gel Extraction Kit and ligated using T4 DNA ligase (Roche, Indianapolis, IN, USA) into pMJ04, which was also digested with the same two restnction enzymes, to generate pMJ05 (Figure 5) Plasmid pMJ05 comprises the Trichoderma reesei cellobiohydrolase I promoter and terminator operably linked to the Humicola insolens endoglucanase V full-length coding sequence

Example 4: Construction of pSMai130 expression vector

A 2586 bp DNA fragment spanning from the ATG start codon to the TAA stop codon of the Aspergillus oryzae beta-glucosidase full-length coding sequence (SEQ ID NO 21 for cDNA sequence and SEQ ID NO 22 for the deduced amino acid sequence, E ∞b DSM 14240) was amplified by PCR from pJaL660 (WO 2002/095014) as template with primers 993467 (sense) and 993456 (antisense) shown below A Spe I site was engineered at the 5' end of the antisense primer to facilitate ligation Primer sequences in italics are homologous to 24 bp of the Trichoderma reesei cbh1 promoter and underlined sequences are homologous to 22 bp of the Aspergillus oryzae beta- glucosidase coding region Primer 993467 δ'-ATAGTCAACCGCGGACTGCGCA ΓCATGAAGCTTGGTTGGATCGAGG-S' (SEQ ID NO 55)

Primer 993456 δ'-ACTAGTTTACTGGGCCTTAGGCAGCG-S (SEQ ID NO 56)

The amplification reactions (50 μl) were composed of Pfx Amplification Buffer 5 (Invitrogen, Carlsbad, CA, USA), 025 mM dNTPs, 10 ng of pJaL660, 64 μM pπmer

993467, 32 μM pπmer 993456, 1 mM MgCI₂, and 2 5 units of Pfx DNA polymerase

(Invitrogen, Carlsbad, CA, USA) The reactions were incubated in an EPPENDORF®

MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94⁰C, 1 minute at 55°C, and 3 minutes at 72⁰C (15 minute final extension) The reaction 10 products were isolated on a 1 0% agarose gel using TAE buffer where a 2586 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction

Kit according to the manufacturer's instructions

A separate PCR was performed to amplify the Tnchoderma reesei cbM promoter sequence extending from 1000 bp upstream of the ATG start codon of the I₅ gene, using primer 993453 (sense) and primer 993463 (antisense) shown below to generate a 1000 bp PCR fragment

Primer 993453 δ'-GTCGACTCGAAGCCCGAATGTAGGAT-S' (SEQ ID NO 57)

Primer 993463 20 5'-CCTCGATCCMCCAAGCπCATGΛTGCGCΛGTCCGCGGrrGACr,A-3' (SEQ ID

NO 58)

Primer sequences in italics are homologous to 24 bp of the Tnchoderma reesei cbM promoter and underlined pπmer sequences are homologous to 22 bp of the Aspergillus oryzae beta-glucosidase full-length coding region The 46 bp overlap between the 25 promoter and the coding sequence allowed precise fusion of the 1000 bp fragment compπsing the Tnchoderma reesei cbM promoter to the 2586 bp fragment compπsing the Aspergillus oryzae beta-glucosidase coding region

The amplification reactions (50 μl) were composed of Pfx Amplification Buffer,

025 mM dNTPs, 100 ng of Tnchoderma reesei RutC30 genomic DNA, 64 μM primer 30 993453, 32 μM primer 993463, 1 mM MgCI₂, and 2 5 units of Pfx DNA polymerase

The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94°C, 1 minute at 55°C, and 3 minutes at 72⁰C (15 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 1000 bp product band was excised from the gel 35 and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

The purified fragments were used as template DNA for subsequent amplification by overlapping PCR using primer 993453 (sense) and primer 993456 (antisense) shown above to precisely fuse the 1000 bp fragment comprising the Trichoderma reesei cbh1 promoter to the 2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase full-length coding region

5 The amplification reactions (50 μl) were composed of Pfx Amplification Buffer,

025 mM dNTPs, 64 μM primer 99353, 32 μM primer 993456, 1 mM MgCI₂, and 25 units of Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94⁰C, 1 minute at 60°C, and 4 minutes at 72°C (15 minute final extension)

10 The resulting 3586 bp fragment was digested with Sal I and Spe I and hgated into pMJ04, digested with the same two restriction enzymes, to generate pSMaι130 (Figure 6) Plasmid pSMaι130 comprises the Trichoderma reesei cellobiohydrolase I gene promoter and terminator operably linked to the Aspergillus oryzae native beta- glucosidase signal sequence and coding sequence (/ e , full-length Aspergillus oryzae

I₅ beta-glucosidase coding sequence)

Example 5: Construction of pSMai135

The Aspergillus oryzae beta-glucosidase mature coding region (minus the native signal sequence, see Figure 7, SEQ ID NOs 59 and 60) from Lys-20 to the TAA stop

20 codon was PCR amplified from pJaL660 as template with pnmer 993728 (sense) and primer 993727 (antisense) shown below Primer 993728

S'-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-S' (SEQ ID NO 61)

2₅ Primer 993727 δ'-GACTAGTCTTACTGGGCCTTAGGCAGCG-S' (SEQ ID NO 62) Sequences in italics are homologous to 20 bp of the Humicola msolens endoglucanase V signal sequence and sequences underlined are homologous to 22 bp of the Aspergillus oryzae beta-glucosidase coding region A Spe I site was engineered into the

30 5' end of the antisense pnmer

The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 025 mM dNTPs, 10 ng/μl of pJaL660, 64 μM pnmer 993728, 32 μM pnmer 993727, 1 mM MgCI₂, and 2 5 units of Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute

35 at 94°C, 1 minute at 55⁰C, and 3 minutes at 72⁰C (15 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 2523 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

A separate PCR amplification was performed to amplify 1000 bp of the Tnchoderma reesei cbM promoter and 63 bp of the Humicola insolens endoglucanase

V signal sequence (ATG start codon to Ala-21 , Figure 8, SEQ ID NOs 63 and 64), using 5 primer 993724 (sense) and primer 9Θ3729 (antisense) shown below

Primer 993724

5'-ACGCGTCGACCGAATGTAGGAπGπATCC-3' (SEQ ID NO 65)

Primer 993729 δ'-GGGAGJACGCGAGAJCAτCCJJGGCAAGGGCCAACACCGGCA-y (SEQ ID NO

10 66)

Pnmer sequences in italics are homologous to 20 bp of the Humicola insolens endoglucanase V signal sequence and underlined pnmer sequences are homologous to the 22 bp of the Aspergillus oryzae beta-glucosidase coding region

Plasmid pMJ05, which comprises the Humicola insolens endoglucanase V

I₅ coding region under the control of the cbM promoter, was used as template to generate a 1063 bp fragment comprising the Tnchoderma reesei cbh1 promoter and Humicola insolens endoglucanase V signal sequence fragment A 42 bp of overlap was shared between the Tnchoderma reesei cbM promoter and Humicola insolens endoglucanase

V signal sequence and the Aspergillus oryzae beta-glucosidase mature coding 20 sequence to provide a perfect linkage between the promoter and the ATG start codon of the 2523 bp Aspergillus oryzae beta-glucosidase coding region

The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 025 mM dNTPs, 10 ng/μl of pMJ05 64 μM pnmer 993728, 32 μM pnmer 993727, 1 mM MgCI₂, and 2 5 units of Pfx DNA polymerase The reactions were incubated in an

25 EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94°C, 1 minute at 6O⁰C, and 4 minutes at 72⁰C (15 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 1063 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

30 The purified overlapping fragments were used as templates for amplification using primer 993724 (sense) and pnmer 993727 (antisense) described above to precisely fuse the 1063 bp fragment comprising the Tnchoderma reesei cbM promoter and Humicola insolens endoglucanase V signal sequence to the 2523 bp fragment compnsing the Aspergillus oryzae beta-glucosidase mature coding region frame by

35 overlapping PCR

The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 025 mM dNTPs, 64 μM primer 993724, 32 μM primer 993727, 1 mM MgCI₂, and 2 5 units of Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94°C, 1 minute at 60°C, and 4 minutes at 72⁰C (15 minute final extension) The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 3591 bp 5 product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

The resulting 3591 bp fragment was digested with Sal I and Spe I and ligated into pMJ04 digested with the same restriction enzymes to generate pSMaιi35 (Figure

9) Plasm id pSMaι135 comprises the Tπchoderma reesei cellobiohydrolase I gene

10 promoter and terminator operably linked to the Humicola msolens endoglucanase V signal sequence and the Aspergillus oryzae beta-glucosidase mature coding sequence

Example 6: Expression of Aspergillus oryzae beta-glucosidase with the Humicola insolens endoglucanase V secretion signal

I₅ Plasmid pSMaι135 encoding the mature Aspergillus oryzae beta-glucosidase linked to the Humicola msolens endoglucanase V secretion signal (Figure 8) was introduced into Trichoderma reesei RutC30 by PEG-mediated transformation (Penttila ef a/ , 1987, Gene 61 155-164) The plasmid contained the Aspergillus mutilans amdS gene to enable transformants to grow on acetamide as the sole nitrogen source

20 Trichoderma reesei RutC30 was cultivated at 27°C and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM undine for 17 hours Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System (Millipore, Bedford, MA, USA) and washed twice with deionized water and twice with 1 2 M sorbitol Protoplasts were generated by suspending the washed mycelia in 20 ml of

25 1 2 M sorbitol containing 15 mg of GLUCANEX® (Novozymes A/S, Bagsvεerd, Denmark) per ml and 036 units of chitinase (Sigma Chemical Co , St Louis, MO, USA) per ml and incubating for 15-25 minutes at 34°C with gentle shaking at 90 rpm Protoplasts were collected by centnfuging for 7 minutes at 400 x g and washed twice with cold 1 2 M sorbitol The protoplasts were counted using a haemacytometer and re-

30 suspended in STC to a final concentration of 1 X 10^s protoplasts per ml Excess protoplasts were stored in a Cryo 1°C Freezing Container (Nalgene, Rochester, NY, USA) at -8O⁰C

Approximately 7 μg of pSMaι135 digested with Pme I was added to 100 μl of protoplast solution and mixed gently, followed by 260 μl of PEG buffer, mixed, and

35 incubated at room temperature for 30 minutes STC (3 ml) was then added and mixed and the transformation solution was plated onto COVE plates using Aspergillus nidulans amdS selection The plates were incubated at 28°C for 5-7 days Transformants were sub-cultured onto COVE2 plates and grown at 28⁰C

Sixty-seven transformants designated SMA135 obtained with pSMaι135 were subcultured onto fresh plates containing acetamide and allowed to sporulate for 7 days at 28°C

5 The 67 SMA135 Tπchoderma reesei transformants were cultivated in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing media at pH 60 inoculated with spores of the transformants and incubated at 28°C and 200 rpm for 7 days Tnchoderma reesei RutC30 was run as a control Culture broth samples were removed at day 7 One ml of each culture broth was centπfuged at 15,700 x g for 5 minutes in a

10 micro-centrifuge and the supematants transferred to new tubes Samples were stored at 4°C until enzyme assay The supematants were assayed for beta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranoside as substrate, as described below

Beta-glucosidase activity was determined at ambient temperature using 25 μl ahquots of culture supematants, diluted 1 10 in 50 mM succinate pH 50, in 200 μl of 05

I₅ mg/ml p-nrtrophenyl-beta-D-glucopyranoside as substrate in 50 mM succinate pH 5 0 After 15 minutes incubation the reaction was stopped by adding 100 μl of 1 M Tπs-HCI pH 80 and the absorbance was read spectrophotometrically at 405 nm One unit of beta-glucosidase activity corresponded to production of 1 μmol of p-nitrophenyl per minute per liter at pH 5 0, ambient temperature Aspergillus niger beta-glucosidase

20 (NOVOZYM ™ 188, Novozymes A/S, Bagsvasrd, Denmark) was used as an enzyme standard

A number of the SMA135 transformants showed beta-glucosidase activities several-fold higher than that secreted by Tnchoderma reesei RutC30 Of the SMA135 transformants screened, transformant SM A135-04 produced the highest beta-

25 glucosidase activity

SDS-PAGE was earned out using CRITERION® Tπs-HCI (5% resolving) gels (Bio-Rad, Hercules, CA, USA) with the CRITERION® System (Bio-Rad, Hercules, CA, USA) Five μl of day 7 supematants (see above) were suspended in 2X concentration of Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and boiled in the presence of

30 5% beta-mercaptoethanol for 3 minutes The supernatant samples were loaded onto a polyacrylamide gel and subjected to electrophoresis with 1X Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, CA, USA) The resulting gel was stained with BIO- SAFE® Coomassie Stain (Bio-Rad, Hercules, CA, USA)

Of the 38 Tnchoderma reesei SMA135 transformants analyzed by SDS-PAGE,

35 26 produced a protein of approximately 110 kDa that was not visible in Tnchoderma reesei RutC30 as control Transformant Tπchoderma reesei SMA135-04 produced the highest level of beta-glucosidase as evidenced by abundance of the 110 kDa band seen by SDS-PAGE

Example 7: Construction of expression vector ρSMai140

Expression vector pSMaι140 was constructed by digesting plasm id pSATe111BG41 (WO 04/099228), which carries the Aspergillus oryzae beta- glucosidase vanant BG41 full-length coding region (SEQ ID NO 23 that encodes the amino acid sequence of SEQ ID NO 24), with Nco I The resulting 1243 bp fragment was isolated on a 1 0% agarose gel using TAE buffer and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions Expression vector ρSMaι135 was digested with Nco I and a 8286 bp fragment was isolated on a 1 0% agarose gel using TAE buffer and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions The 1243 bp Nco I digested Aspergillus oryzae beta-glucosidase vanant BG41 fragment was then ligated to the 8286 bp vector, using T4 DNA ligase (Roche, Indianapolis, IN, USA) according to manufacturer's protocol, to create the expression vector pSMaι140 (Figure 10) Plasm id ρSMaι140 compnses the Tnchodemna reesei cellobiohydrolase I (CEL7A) gene promoter and terminator operably linked to the Humicola insolens endoglucanase V signal sequence and the Aspergillus oryzae beta-glucosidase vanant mature coding sequence

Example 8: Transformation of Trichoderma reesei RutC30 with ρSMai140

Plasmid pSMaι140 was linearized with Pme I and transformed into the Trichoderma reesei RutC30 strain as described in Example 6 A total of 100 transformants were obtained from four independent transformation expeπments, all of which were cultivated in shake flasks on cellulase-inducing medium, and the beta- glucosidase activity was measured from the culture medium of the transformants as described in Example 6 A number of Trichoderma reesei SMA140 transformants showed beta-glucosidase activities several fold higher than that of Trichoderma reesei RutC30 The presence of the Aspergillus oryzae beta-glucosidase variant BG41 protein in the culture medium was detected by SDS-polyacrylamide gel electrophoresis as described in Example 6 and Coomassie staining from the same 13 culture supernatants from which enzyme activity were analyzed All thirteen transformants that had high β- glucosidase activity, also expressed the approximately 110 KDa Aspergillus oryzae beta-glucosidase variant BG41 , at varying yields

The highest beta-glucosidase vanant expressing transformant, as evaluated by beta-glucosidase activity assay and SDS-polyacrylamide gel electrophoresis, was designated Tnchoderma reesei SMA140-43

Example 9: Construction of expression vector ρSaMe-F1

A DNA fragment containing 209 bp of the Tnchoderma reesei cellobiohydrolase I 5 gene promoter and the core region (nucleotides 1 to 702 of SEQ ID NO 1 that encode amino acids 1 to 234 of SEQ ID NO 2, WO 91/17243) of the Humi∞la insolens endoglucanase V gene was PCR amplified using pMJ05 as template using the primers shown below

995103 10 5'-cccaagcttagccaagaaca-3' (SEQ ID NO 67)

995137

5'-gggggaggaacgcatgggatctggacggc-3' (SEQ ID NO 68)

The amplification reactions (50 μl) were composed of 1X Pfx Amplification

Buffer, 10 mM dNTPs, 50 mM MgSO₄, 10 ng/μl of pMJ05, 50 picomoles of 995103 I₅ primer, 50 picomoles of 995137 primer, and 2 units of Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94°C, 30 seconds at 55°C, and 60 seconds at 72°C

(3 minute final extension)

The reaction products were isolated on a 1 0% agarose gel using TAE buffer 20 where a 911 bp product band was excised from the gel and purified using a

QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

A DNA fragment containing 806 bp of the Aspergillus oryzae beta-glucosidase vaπant BG41 gene was PCR amplified using pSMaι140 as template and the primers shown below 2₅ 995133

5'-gccgtccagatccccatgcgttcctccccc-3' (SEQ ID NO 69)

995111

5'-ccaagcttgttcagagtttc-3' (SEQ ID NO 70)

The amplification reactions (50 μl) were composed of 1X Pfx Amplification 30 Buffer, 10 mM dNTPs, 50 mM MgSO₄, 100 ng of pSMaι140, 50 picomoles of 995133 primer, 50 picomoles of 995111 primer, and 2 units of Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94°C, 30 seconds at 55°C, and 120 seconds at

72°C (3 minute final extension) 35 The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 806 bp product band was excised from the gel and purified using a

QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions The two PCR fragments above were then subjected to overlapping PCR The purified overlapping fragments were used as templates for amplification using pπmer 995103 (sense) and primer 995111 (antisense) described above to precisely fuse the 702 bp fragment comprising 209 bp of the Tnchoderma reesei cellobiohydrolase I gene promoter and the Humicola insolens endoglucanase V core sequence to the 806 bp fragment compnsing a portion of the Aspergillus oryzae beta-glucosidase variant BG41 coding region by overlapping PCR

The amplification reactions (50 μl) were composed of 1X Pfx Amplification Buffer, 10 mM dNTPs, 50 mM MgSO₄, 25 μl of each fragment (20 ng/μl), 50 picomoles of 995103 primer, 50 picomoles of 995111 pπmer and 2 units of high fidelity Pfx DNA polymerase The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for an initial denaturation of 3 minutes at 95°C followed by 30 cycles each for 1 minute of denaturation, 1 minute annealing at 60⁰C, and a 3 minute extension at 72°C The reaction products were isolated on a 1 0% agarose gel using TAE buffer where a 1 7 kb product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions

The 1 7 kb fragment was ligated into a pCR®4 Blunt Vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions The construct was then transformed into ONE SHOT® TOP10 Chemically Competent E coli cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's rapid chemical transformation procedure Colonies were selected and analyzed by plasmid isolation and digestion with Hind III to release the 1 7 kb overlapping PCR fragment

Plasmid pSMaι140 was also digested with Hind III to linearize the plasmid Both digested fragments were combined in a ligation reaction using a Rapid DNA Ligation Kit following the manufacturer's instructions to produce pSaMe-F1 (Figure 11)

E coli XL1-Blue Subcloning-Grade Competent Cells (Stratagene, La JoIIa, CA, USA) were transformed with the ligation product Identity of the construct was confirmed by DNA sequencing of the Tnchoderma reesei cellobiohydrolase I gene promoter Humicola insolens endoglucanase V signal sequence, Humicola msolens endoglucanase V core, Humicola insolens endoglucanase V signal sequence Aspergillus oryzae beta-glucosidase vanant BG41, and the Tnchoderma reesei cellobiohydrolase I gene terminator sequence from plasmids purified from transformed E coli One clone containing the recombinant plasmid was designated pSaMe-F1 Plasmid pSaMe-F1 comprises the Tnchoderma reesei cellobiohydrolase I gene promoter and terminator and the Humicola insolens endoglucanase V signal peptide sequence linked directly to the Humicola insolens endoglucanase V core polypeptide that are fused directly to the Humicola insolens eπdoglucaπase V signal peptide that is linked directly to the Aspergillus oryzae beta-glucosidase variant BG41 mature coding sequence The DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase vaπant BG fusion protein is shown in SEQ ID NOs 73 and 74, respectively (see Figures 14A, 14B, 14C, and 14D)

Example 10: Transformation of Trichoderma reesei RutC30 with pSaMe-F1

Shake flasks containing 25 ml of YP medium supplemented with 2% glucose and 10 mM undine were inoculated with 5 X 10⁷ spores of Tnchoderma reesei RutC30 Following incubation overnight for approximately 16 hours at 27⁰C, 90 rpm, the mycelia were collected using a Vacuum Driven Disposable Filtration System The mycelia were washed twice in 100 ml of deiomzed water and twice in 1 2 M sorbitol Protoplasts were generated as described in Example 6

Two micrograms of pSaMe-F1 DNA linearized with Pme 1, 100 μl of Trichoderma reesei RutC30 protoplasts, and 50% PEG (4000) were mixed and incubated for 30 minutes at room temperature Then 3 ml of STC were added and the contents were poured onto a COVE plate supplemented with 10 mM undine The plate was then incubated at 28°C Transformants began to appear by day 6 and were picked to COVE2 plates for growth at 28°C and 6 days Twenty-two Trichoderma reesei transformants were recovered

Transformants were cultivated in shake flasks on cellulase-inducing medium, and beta-glucosidase activity was measured as described in Example 6 A number of pSaMe-F1 transformants produced beta-glucosidase activity One transformant, designated Trichoderma reesei SaMeF1-9, produced the highest amount of beta- glucosidase, and had twice the activity of a strain expressing the Aspergillus oryzae beta-glucosidase variant (Example 9)

Endoglucanase activity was assayed using a carboxymethyl cellulose (CMC) overlay assay according to Begum, 1983, Analytical Biochem 131(2) 333-336 Five μg of total protein from five of the broth samples (those having the highest beta-glucosidase activity) were diluted in Native Sample Buffer (Bio-Rad, Hercules, CA, USA) and run on a CRITERION® 8-16% Tπs-HCI gel (Bio-Rad, Hercules, CA, USA) using 10X Tris/glycine running buffer (Bio-Rad, Hercules, CA, USA) and then the gel was laid on top of a plate containing 1% carboxymethylcellulose (CMC) After 1 hour incubation at 37°C, the gel was stained with 0 1% Congo Red for 20 minutes The plate was then destained using 1 M NaCI in order to identify regions of clearing indicative of endoglucanase activity Two cleanng zones were visible, one upper zone at approximately 110 kDa and a lower zone at approximately 25 kDa The predicted protein size of the Humicola insolens endoglucanase V and Aspergillus oryzae beta- glucosidase variant BG41 fusion is 118 kDa if the two proteins are not cleaved and remain as a single polypeptide, glycosylation of the individual endoglucanase V core domain and of the beta-glucosidase leads to migration of the individual proteins at higher mw than predicted from the primary sequence If the two proteins are cleaved then the predicted sizes for the Humicola insolens endoglucanase V core domain is 24 kDa and 94 kDa for Aspergillus oryzae beta-glucosidase variant BG41 Since there was a cleanng zone at approximately 110 kDa this result indicated that minimally a population of the endoglucanase and beta-glucosidase fusion protein remains intact as a single large protein The lower cleanng zone most likely represents an endogenous endoglucanase activity, and possibly additionally results from partial cleavage of the Humicola insotens endoglucanase V core domain from the Aspergillus oryzae β- glucosidase

The results demonstrated the Humicola insolens endoglucanase V core was active even while fused to the Aspergillus oryzae beta-glucosidase In addition, the increase in beta-glucosidase activity appeared to result from increased secretion of protein relative to the secretion efficiency of the non-fusion beta-glucosidase ESy linking the Aspergillus oryzae beta-glucosidase variant BG41 sequence to the efficiently secreted Humicola insolens endoglucanase V core, more beta-glucosidase was secreted

Example 11: Construction of vector pSaMe-FX

Plasmid pSaMe-FX was constructed by modifying pSaMe-F1 Plasmid pSaMe- F1 was digested with Bst Z17 and Eco Rl to generate a 1 kb fragment that contained the beta-glucosidase vaπant BG41 coding sequence and a 9 2 kb fragment containing the remainder of the plasmid The fragments were separated on a 1 0% agarose gel using TAE buffer and the 9 2 kb fragment was excised and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions Plasmid pSMaι135 was also digested with Bst Z17 and Eco Rl to generate a 1 kb fragment containing bases homologous to the Aspergillus oryzae beta-glucosidase vaπant BG41 coding sequence and a 8 5 kb fragment containing the remainder of the plasmid The 1 kb fragment was isolated and purified as above

The 9 2 kb and 1 kb fragments were combined in a ligation reaction using a Rapid DNA Ligation Kit following the manufacturer's instructions to produce pSaMe-FX, which is identical to pSaMe-F1 except that it contained the wild-type beta-glucosidase mature coding sequence rather than the vaπant mature coding sequence

E co// SURE® Competent Cells (Stratagene, La JoIIa, CA, USA) were transformed with the ligation product Identity of the construct was confirmed by DNA sequencing of the Tπchoderma reesei cellobiohydrolase I gene promoter, Humicola insolens endoglucanase V signal sequence, Humicoia insolens endoglucanase V core sequence, Humicola insolens endoglucanase V signal sequence, Aspergillus oryzae beta-glucosidase mature coding sequence, and the Tnchoderma reesei cellobiohydrolase I gene terminator sequence from plasmids puπfied from transformed E coli One clone containing the recombinant plasmid was designated pSaMe-FX (Figure 12) The DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase fusion protein is shown in SEQ ID NOs 75 and 76, respectively (see Figures 15A, 15B, 15C, and 15D)

Example 12: Transformation and expression of Trichoderma transformants

The pSaMe-FX construct was linearized with Pme I and transformed into the Trichoderma reesei RutC30 strain as described in Example 10 A total of 63 transformants were obtained from a single transformation Transformants were cultivated in shake flasks on cellulase-inducing medium, and beta-glucosidase activity was measured as described in Example 6 A number of pSaMe-FX transformants produced beta-glucosidase activity One transformant designated SaMe-FXI 6 produced twice the amount of beta-glucosidase activity compared to Tπchoderma reesei SaMeFI -9 (Example 10)

Example 13: Analysis of Trichoderma reesei transformants

A fusion protein was constructed as described in Example 9 by fusing the Humicola insolens endoglucanase V core (containing its own native signal sequence) with the Aspergillus oryzae beta-glucosidase variant BG41 mature coding sequence linked to the Humicola insolens endoglucanase V signal sequence This fusion construct resulted in a two-fold increase in secreted beta-glucosidase activity compared to the Aspergillus oryzae beta-glucosidase vanant BG41 mature coding sequence linked to the Humicola insolens endoglucanase V signal sequence A second fusion construct was made as described in Example 11 consisting of the Humicola insolens endoglucanase V core (containing its own signal sequence) fused with the Aspergillus oryzae wild-type beta-glucosidase coding sequence linked to the Humicola insolens endoglucanase V signal sequence, and this led to an even further improvement in beta- glucosidase activity The strain transformed with the wild-type fusion had twice the secreted beta-glucosidase activity relative to the strain transformed with the beta- glucosidase variant BG41 fusion

Example 14: Cloning of the beta-glucosidase fusion protein encoding sequence into an Aspergillus oryzae expression vector

Two synthetic oligonucleotide primers, shown below, were designed to PCR amplify the full-length open reading frame from pSaMeFX encoding the beta- glucosidase fusion protein PCR Forward primer δ'-GGACTGCGCAGCATGCGTTC-S' (SEQ ID NO 71)

PCR Reverse pnmer

5'-AGTTAATTAATTACTGGGCCTTAGGCAGCG-S' (SEQ ID NO 72)

Bold letters represent coding sequence The underlined "G" in the forward primer represents a base change introduced to create an Sph I restriction site The remaining sequence contains sequence identity compared with the insertion sites of pSaMeFX The underlined sequence in the reverse primer represents a Pac I restriction site added to facilitate the cloning of this gene in the expression vector pAILo2 (WO 04/099228)

Fifty picomoles of each of the primers above were used in a PCR reaction containing 50 ng of pSaMeFX DNA, 1X Pfx Amplification Buffer, 6 μJ of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 25 units of PLATINUM® Pfx DNA Polymerase, and 1 μl of 50 mM MgSO₄ in a final volume of 50 μl An EPPENDORF® MASTERCYCLER® 5333 was used to amplify the fragment programmed for 1 cycle at 98°C for 2 minutes, and 35 cycles each at 96°C for 30 seconds, 61⁰C for 30 seconds, and 68⁰C for 3 minutes After the 35 cycles, the reaction was incubated at 68⁰C for 10 minutes and then cooled at 10°C until further processed A 33 kb PCR reaction product was isolated on a 08% GTG-agarose gel (Cambrex Byproducts One Meadowlands Plaza East Rutherford, NJ, USA) using TAE buffer and 0 1 μg of ethidium bromide per ml The DNA was visualized with the aid of a DARK READER™ (Clare Chemical Research, Dolores, CO, USA) to avoid UV-ιnduced mutations A 3 3 kb DNA band was excised with a disposable razor blade and purified with an ULTRAFREE®-DA spin cup (Millipore, Billeπca, MA, USA) according to the manufacturer's instructions

The purified 33 kb PCR product was cloned into a pCR®4Blunt-TOPO® vector (Invitrogen, Carlsbad, CA, USA) Four microliters of the purified PCR product were mixed with 1 μl of a 2 M sodium chloride solution and 1 μl of the TOPO® vector The reaction was incubated at room temperarature for 15 minutes and then 2 μl of the reaction were used to transform One Shot® TOP10 Chemically Competent E coli cells according to the manufacturer's instructions Three ahquots of 83 μl each of the transformation reaction were spread onto three 150 mm 2X YT plates supplemented with 100 μg of ampicillin per ml and incubated overnight at 37⁰C

Eight recombinant colonies were used to inoculate liquid cultures containing 3 ml of LB medium supplemented with 100 μg of ampicillin per ml Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600 (QIAGEN Iπc , Valencia, CA, USA) Clones were analyzed by restriction enzyme digestion with Pac I Plasmid DNA from each clone was digested with Pac I and analyzed by 1 0% agarose gel electrophoresis using TAE buffer All eight clones had the expected restriction digest pattern and clones 5, 6, 7, and 8 were selected to be sequenced to confirm that there were no mutations in the cloned insert Sequence analysis of their 5' and 3' ends indicated that all 4 clones had the correct sequence Clones 5 and 7 were selected for further sequencing Both clones were sequenced to Phred Q values of greater than 40 to ensure that there were no PCR induced errors Clones 5 and 7 were shown to have the expected sequence and clone 5 was selected for re-cloning into ρAILo2

Plasmid DNA from clone 5 was linearized by digestion with Sph I The linearized clone was then blunt-ended by adding 1 2 μl of a 10 mM blend of dATP, dTTP, dGTP, and dCTP and 6 units of T4 DNA polymerase (New England Bioloabs, lnc , Ipswich, MA, USA) The mixture was incubated at 12°C for 20 minutes and then the reaction was stopped by adding 1 μl of 0 5 M EDTA and heating at 75°C for 20 minutes to inactivate the enzyme A 3 3 kb fragment encoding the beta-glucosidase fusion protein was purified by gel electrophoresis and ultrafiltration as descnbed above

The vector pAILo2 was linearized by digestion with Λfco I The linearized vector was then blunt-ended by adding 0 5 μl of a 10 mM blend of dATP, dTTP, dGTP, and dCTP and one unit of DNA polymerase I The mixture was incubated at 25°C for 15 minutes and then the reaction was stopped by adding 1 μl of 0 5M EDTA and heating at 75°C for 15 minutes to inactivate the enzymes Then the vector was digested with Pac I The blunt-ended vector was purified by gel electrophoresis and ultrafiltration as described above Cloning of the 3 3 kb fragment encoding the beta-glucosidase fusion protein into the linearized and purified pAILo2 vector was performed with a Rapid Ligation Kit A 1 μl sample of the reaction was used to transform E ∞li XL10 SOLOPACK* Gold cells (Stratagene, La JoIIa, CA, USA) according to the manufacturer's instructions After the recovery period, two 100 μl ahquots from the transformation reaction were plated onto two 150 mm 2X YT plates supplemented with 100 μg of ampicillin per ml and incubated overnight at 37⁰C A set of eight putative recombinant clones was selected at random from the selection plates and plasmid DNA was prepared from each one using a BIOROBOT® 9600 Clones 1-4 were selected for sequencing with pAILo2-specιfic primers to confirm that the junction vector/insert had the correct sequence Clone 3 had a perfect vector/insert junction and was designated pAILo47 (Figure 13)

In order to create a marker-free expression strain, a restriction endonuclease digestion was performed to separate the blaA gene that confers resistance to the antibiotic ampicilhπ from the rest of the expression construct Thirty micrograms of pAILo47 were digested with Pme I The digested DNA was then purified by agarose gel electrophoresis as descnbed above A 64 kb DNA band containing the expression construct but lacking the blaA gene was excised with a razor blade and purified with a QIAQUICK® Gel Extraction Kit

Example 15: Expression of the beta-glucosidase fusion protein in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 00/240694) protoplasts were prepared according to the method of Christensen et al , 1988, Bio/Technology 6 1419-1422 Ten microliters of the purified expression construct of Example 14 were used to transform Aspergillus oryzae JaL355 protoplasts The transformation of Aspergillus oryzae JaL355 yielded approximately 90 transformants Fifty transformants were isolated to individual PDA plates and incubated for five days at 34⁰C Forty-eight confluent spore plates were washed with 3 ml of 0 01% TWEEN® 80 and the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glass shake flasks Transformant cultures were incubated at 34⁰C with constant shaking at 200 rpm After 5 days, 1 ml aliquots of each culture was centπfuged at 12,000 x g and their supernatants collected Five μl of each supernatant were mixed with an equal volume of 2X loading buffer (10% beta-mercaptoethanol) and loaded onto a 1 5 mm 8 %-16 % Tns-Glycine SDS-PAGE gel and stained with BIO-SAFE® Coomassie Blue G250 protein stain (Bio-Rad, Hercules, CA, USA) SDS-PAGE profiles of the culture broths showed that 33 out of 48 transformants were capable of expressing a new protein with an apparent molecular weight very close to the expected 118 kDa Transformant 21 produced the best yield and was selected for further studies

Example 16: Single Spore Isolation of Aspergillus oryzae JaL355 Transformant 21

Aspergillus oryzae JaL355 transformant 21 spores were spread onto a PDA plate and incubated for five days at 34⁰C A small area of the confluent spore plate was washed with 0 5 ml of 001% TWEEN® 80 to resuspend the spores A 100 μl aliquot of the spore suspension was diluted to a final volume of 5 ml with 001% TWEEN® 80 With the aid of a hemocytometer the spore concentration was determined and diluted to a final concentration of 0 1 spores per microliter A 200 μl aliquot of the spore dilution was spread onto 150 mm Minimal medium plates and incubated for 2-3 days at 34⁰C Emerging colonies were excised from the plates and transferred to PDA plates and incubated for 3 days at 34⁰C Then the spores were spread across the plates and incubated again for 5 days at 34⁰C The confluent spore plates were washed with 3 ml of 0 01% TWEEN® 80 and the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glass shake flasks Single-spore cultures were incubated at 34⁰C with constant shaking at 200 rpm After 5 days, a 1 ml aliquot of each culture was centrifuged at 12,000 x g and their supernatants collected Five μl of each supernatant were mixed with an equal volume of 2X loading buffer (10% beta-mercaptoethanol) and loaded onto a 1 5 mm 8%- 16% Tπs-Glycine SDS-PAGE gel and stained with BIO-SAFE® Commassie Blue G250 protein stain SDS-PAGE profiles of the culture broths showed that all eight transformants were capable of expressing the beta-glucosidase fusion protein at very high levels and one of the cultures designated Aspergillus oryzae JaL355AILo47 produced the best yield

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention Any equivalent aspects are intended to be within the scope of this invention Indeed, vanous modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description Such modifications are also intended to fall within the scope of the appended claims In the case of conflict, the present disclosure including definitions will control

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties

Claims

What ts claimed Is:

5 1. A method for producing a secreted poiypeptide having biological activity, comprising:

(a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, wherein the fusion protein construct comprises;

(i) a first polynucleotide comprising a nucleotide sequence encoding K) a signal peptide;

(it) a second poiynucleotide comprising a nucleotide sequence encoding at feast a catalytic domain of an endoglucanase or a portion thereof; and

(iii) a third polynucleotide comprising a nucleotide sequence encoding 15 at least a catalytic domain of a polypeptide having biological activity or a portion thereof; wherein the signal peptide and at least the catalytic domain of the endoglucanase or the portion thereof increases secretion of the polypeptide having biological activity or the portion thereof compared to the absence of at 20 least the catalytic domain of the endoglucanase or the portion thereof;

(b) activating the transformed fungal host cell under conditions suitable for production of the fusion protein; and

(c) recoveήng the fusion protein, a component thereof, or a combination thereof, from the cultivation medium, wherein the fusion protein or the component 5 thereof has biological activity.

2. The method of claim 1 , wherein the 3' end of the ftrst polynucleotide is operably linked to the 5' end of the second polynucleotide and the 3' end of the second polynucleotide is operably linked to the 5' end of the third polynucleotide or the 3' end

30 of the first polynucleotide is operably linked to the 5' end of the third polynucleotide and the 3^r end of the third polynucleotide is operabfy linked to the 5^r end of the second polynucleotide to encode a fusion protein,

3. The method of claim 1 or 2, wherein the first polynucleotide encoding the signal 35 peptide is obtained from a Humicola insolens CEL45 endogiucanase gene (cel45).

4. The method of any of claims 1-3, wherein the second polynucleotide comprising the nucleotide sequence encoding at least the endoglucanase catalytic domain or the portion thereof is obtained from a Humicola insolens CEL45 endogSucanase gene (ce/45).

5, The method of any of claims 1-3, wherein the second polynucleotide comprising the nucleotide sequence encoding at least the endoglucanase catalytic domain or the portion thereof is obtained from a gene encoding an endoglucanase comprising an amino acid sequence having at Seast 70%, preferabSy at ieast 75%, more preferably at Sβast 80%, even more preferably at ieast 85%, most preferably at least 90%, and even most preferably at Seast 95% identity to SEQ ID NO: 2.

8, The method of any of claims 1-5, wherein the fusion protein construct further comprises a polynucieotide encoding a carbohydrate binding moduie.

7. The method of any of claims 1-8, wherein the second polynucleotide encodes a catalytic domain or a portion thereof, a mature polypeptide, or a fuli-iength polypeptide of the endoglucanase.

8, The methods of any of ciaims 1-7, wherein the third polynucleotide encodes a catalytic domain or a portion thereof, a mature polypeptide, or a fuli-iength polypeptide of the polypeptide having bioiogiεal activity,

9, The method of any of claims 1-8, wherein the fusion protein construct further comprises a polynucleotide encoding a Sinker located between the nucleotide sequence encoding at ieast the endogiucanase catalytic domain or the portion thereof and the nucleotide sequence encoding at ieast the catalytic domain of a polypeptide having biologicai activity or the portion thereof and alternatively also a polynucleotide encoding a cleavage site located between the nucleotide sequence encoding at least the endoglucanase catalytic domain or the portion thereof and the nucleotide sequence encoding at least the catalytic domain of a polypeptide having biological activity or the portion thereof.

10, The method of any of claims 1-9, wherein the fusion protein construct further comprises a fourth polynucleotide encoding a second signal peptide.

11 The method of claim 10, wherein the 3' enά of the fourth polynucleotide is operabiy linked to the 5¹ end of the second polynucleotide or the third polynucleotide.

12. The method of any of claims 1-11 , wherein the fusion protein construct further comprises a promoter and/or a terminator.

5 13, The method of claim 12, wherein the promoter drives expression of the first, second, and third polynucleotides, and alternatively also the fourth polynucleotide.

14. The method of any of claims 1-13, wherein the polypeptide having biological activity is an antibody, antigen, antimicrobia! peptide, enzyme, growth factor, hormone,

H) irnmunodiSator, neurotransmitter, receptor, reporter protein, or structural protein,

15, The method of any of claims 1-14, wherein the polypeptide having biological activity is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or iigase,

15 16. The method of any of claims 1-15, wherein the polypeptide having biological activity is a ceiluloSytic enzyme or a hemiceliulolytic enzyme.

17, The method of claim 16, wherein the ceiluloiytic enzyme is a beta-giucosidase.

20 18. The method of claim 17, wherein the beta-glucosidase comprises an amino acid sequence having at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to SEQ ID NO: 24 or a variant beta-glucosidase comprising an amino acid sequence having at least 70%, preferably at least 75%, 5 more preferably at least 80%, even more preferably at least 85%, most preferably at ieast 90%, and even most preferably at least 95% identity to SEQ ID NO; 35,

19. The method of any of claims 1-18, wherein the fusion protein, a component thereof, or a combination thereof, has celluioiytic activity or hemsceliuiolytic activity,

30

20. An isolated fusion protein, comprising:

(a) a first amino acid sequence comprising a signal peptide;

(b) a second amino acid sequence comprising at ieast a catalytic domain of an endoglucanase or a portion thereof; and

35 (c) a third amino acid sequence comprising at least a catalytic domain of a polypeptide having bioiogicai activity or a portion thereof.

21. The fusion protein of claim 20, wherein the C-terminai end of the first amino acid sequence is linked in frame to the M-terminal end of the second amino acid sequence and the C-terminal end of the second amino acid sequence is linked in frame to the N- ierminal enύ of the third amino acid sequence or the C-terminai enύ of the first amino

5 acid sequence is linked in frame to the N-terminaS end of the third amino acid sequence and the C-terminai end of the third amino acid sequence is iinked in frame to the N- terminai end of the second amino acid sequence,

22. The fusion protein of claim 20 or 21 , wherein the first amino acid sequence H) comprising the signal peptide is obtained from Hυmicola insolens CEL45 endogiueanase.

23. The fusion protein of any of claims 20-22, wherein the second amino acid sequence comprising at Seast the endogiucanase catalytic domain is obtained from a

15 Humicota ϊnsolens CEL45 endogiucanase.

24. The fusion protein of any of claims 20-22« wherein the second amino acid sequence comprising at ieast the endogiucanase catalytic domain is obtained from an endogiucanase comprising an amino acid sequence having at ieast 70%, preferably at

20 ieast 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, anά even most preferably at least 95% identity to SEQ ID NO: 2.

25. The fusion protein of any of claims 20-24, which further comprises a 5 carbohydrate binding module,

26. The fusion protein of any of claims 20-25, wherein the second amino acid sequence comprises a catalytic domain or a portion thereof, a mature polypeptide, or a full-length polypeptide of the endogiucanase,

30

27. The fusion protein of any of claims 20-26, wherein the third amino acid sequence comprises a catalytic domain or a portion thereof, a mature polypeptide, or a full-length polypeptide of the polypeptide having biological activity.

35 28. The fusion protein of any of claims 20-27, which further comprises a linker located between at least the endogiucanase catalytic domain or the portion thereof and at least the catalytic domain of the polypeptide having biological activity or the portion thereof and alternatively also a cleavage site located between at least the endogiucanase catalytic domain or the portion thereof and at least the catalytic domain of a polypeptide having biological activity or the portion thereof.

5 29. The fusion protein of any of claims 20-28, which further comprises a fourth amino acid sequence comprising a second signai peptide.

30, The fusion protein of any of claims 20-29, wherein the second signal peptide is linked in frame to the N-terminus of the amino acid sequence comprising at least the

H) catalytic domain of the polypeptide having biological activity or the portion thereof or at ieast the catalytic domain of the endogiucanase or the portion thereof,

31. The fusion protein of any of claims 20-30, wherein the polypeptide having biological activity is an antibody, antigen, antimicrobia! peptide, enzyme, growth factor,

15 hormone, immunodiiator, neurotransmitter, receptor, reporter protein, or structural protein,

32. The fusion protein of any of claims 20-31 , wherein the polypeptide having biological activity is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or

20 ligase.

33, The fusion protein of any of ciaSms 20-32, wherein the polypeptide having biological activity is a ceilulolytic enzyme or a hemicellulolytic enzyme, 5 34. The fusion protein of claim 33, wherein the ceiluloiytic enzyme is a beta- glυcosidase,

35, The fusion protein of claim 34, wherein the beta-glucosidase comprises an amino acid sequence having at ieast 70%, preferably at least 75%, more preferably at

30 least 80%, even more preferably at ieast 85%, most preferably at ieast 90%, and even most preferably at least 95% identity to SEQ ID NO; 24 or a variant beta-glucosidase comprising an amino acid sequence having at least 70%, preferably at least 75%, more preferably at least 80%, even more preferabiy at least 85%, most preferably at least 90%, and even most preferably at ieast 95% identity to SEQ SD NO: 35,

35

36. The fusion protein of claim 34, wherein the beta-gSucosidase is SEQ ID NO; 24 or SEQ ID NO; 35.

37. The fusion protein of any of ciairns 20-36, which is a faeta-giucosidase fusion protein or components thereof.

5 38. The fusion protein of claim 37, which comprises SEQ ID NO: 74 or SEQ SD NO; 76.

39, The fusion protein of cSaim 38_S wherein SEQ ID NO: 74 is encoded by SEQ ID NO: 73 and SEQ ID NO: 76 is encoded by SEQ ID NO: 75.

H⁾

40. The fusion protein of any of claims 20-40 wherein the fusion protein, a component thereof, or a combination thereof, has ceiiulolytic activity or hemiceiluloiytic activity

15 41. An isolated polynucleotide encoding the fusion protein of any of ciairns 20-40.

42. A fusion protein construct, comprising the polynucleotide of claim 41 ,

43. An expression vector, comprising the fusion protein construct of claim 42. 20

44. A fungal host ceil transformed with the fusion protein construct of claim 43.

45. A method for degrading or converting a celiulosic materia!, comprising: treating the celiulosic material with an effective amount of a ceiluloiytic enzyme composition in 5 the presence of an effective amount of the fusion protein of claim 40 or a component thereof,

46. The method of claim 45, wherein the celiuiolytic enzyme composition comprises one or more celSulolytic proteins are selected from the group consisting of a celiuiase, an

30 endogiucanase, a ceilobionydroiase, and a polypeptide having celiuiolytic enhancing activity.

47. The method of claim 45 or 46, further comprising treating the celiulosic material with an effective amount of one or more enzymes selected from the group consisting of

35 a hemicellulase, esterase, protease, iaccase, peroxidase, or a mixture thereof.

48. The method of any of claims 45-47, wherein the method is a pretreatment process, a step in a simultaneous saccharification and fermentation process (SSF), a step in a separate hydrolysis anά fermentation (SHF), or a step in a hybrid hydrolysis and fermentation process (HHF).

5 49. The method of any of claims 45-48. further comprising recovering the degraded ceilulosic material.

50. The method of claim 45, wherein the ceSlulolytic protein and/or fusion protein or the component thereof are in the form of a fermentation broth with or without cells.

H⁾

51. A method for producing a substance, comprising:

(a) saccharifying a celSυiosic materia! with an effective amount of a ceiluloiytic enzyme composition in the presence of an effective amount of the fusion protein of claim 40 or a component thereof;

15 (b) fermenting the saccharified celluSosic material of step (a) with one or more fermenting microorganisms; and

(C) recovering the organic substance from the fermentation,

52. The method of claim 51 , wherein the celiuiolytic enzyme composition comprises 20 one or more proteins are selected from the group consisting of a celiuiase, an endogiucanase, a ceilobiohydroiase, and a polypeptide having celluioiytic enhancing activity.

53. The method of cSaim 51 or 52, further comprising treating the cellυiosic material 5 with an effective amount of one or more enzymes selected from the group consisting of a hemicelluiase, esterase, protease, laccase. peroxidase, or a mixture thereof.

54. The method of any of claims 51-53, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharificatSon and fermentation.

30

55. The method of any of claims 51-53, wherein the substance is an aicohol, organic acid, ketone, amino acid, or gas,

56. The method of any of claims 51-55, wherein the celluioiytic enzyme composition 35 and/or the fusion protein or a component thereof are in the form of a fermentation broth with or without cells.