Exo-endo cellulase fusion protein

Abstract

The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

1 . A heterologous exo-endo cellulase fusion construct comprising in operable linkage from the 5′ end of said molecule
 a. a DNA molecule encoding a signal sequence; 
 b. a DNA molecule encoding a catalytic domain of an exo-cellobiohydrolase; and 
 c. a DNA molecule encoding an endoglucanase catalytic domain. 
 
     
     
         2 . The heterologous exo-endo cellulase fusion construct according to  claim 1  further comprising a linker sequence located 3′ of the catalytic domain of the exo-cellobiohydrolase and 5′ of the catalytic domain of the endoglucanase. 
     
     
         3 . The heterologous exo-endo cellulase fusion construct according to  claim 1  further comprising a kexin site located after the linker sequence and before the coding region of the endoglucanase catalytic domain. 
     
     
         4 . The heterologous exo-endo fusion construct according to  claim 1  further comprising a promoter of a filamentous fungus secretable protein, said promoter located in operable linkage 5′ of the coding region of the exo-cellobiohydrolase catalytic domain. 
     
     
         5 . The heterologous exo-endo fusion construct according to  claim 4  wherein the promoter is a cbh promoter. 
     
     
         6 . The heterologous exo-endo fusion construct according to  claim 5  wherein the promoter is a cbh1 promoter derived from  T. reesei.    
     
     
         7 . The heterologous exo-endo fusion construct according to  claim 1  wherein the exo-cellobiohydrolase is a CBH1. 
     
     
         8 . The heterologous exo-endo fusion construct according to  claim 7  wherein said CBH1 comprises an amino acid sequence of at least 90% sequence identity with the sequence set forth in SEQ ID NO.: 6. 
     
     
         9 . The heterologous exo-endo fusion construct according to  claim 1  wherein the endoglucanase catalytic domain is derived from a bacterial endoglucanase. 
     
     
         10 . The heterologous exo-endo fusion construct according to  claim 9  wherein the bacterial endoglucanase catalytic domain is selected from the group consisting of an  Acidothermus cellulolyticus  GH5A endoglucanase I (E1) catalytic domain; an  Acidothermus cellulolyticus  GH74 endoglucanase (GH74-EG) catalytic domain: and a  Thermobifida fusca  E5 endoglucanase (Tf-E5) catalytic domain. 
     
     
         11 . The heterologous exo-endo fusion construct according to  claim 10  wherein the endoglucanase is an  Acidothermus cellulolyticus  GH5A E1 catalytic domain. 
     
     
         12 . The heterologous exo-endo fusion construct according to  claim 10  wherein the  Acidothermus cellulolyticus  GH5A E1 catalytic domain having an amino acid sequence of at least 90% sequence identity with the sequence set forth in SEQ ID NO. 8. 
     
     
         13 . The heterologous exo-endo fusion construct according to  claim 1  further comprising a terminator sequence located 3′ to the endoglucanase catalytic domain. 
     
     
         14 . The heterologous exo-endo fusion construct according to  claim 1  further comprising a selectable marker. 
     
     
         15 . A vector comprising in operable linkage a promoter of a filamentous fungus secretable protein, a DNA molecule encoding a signal sequence, a DNA molecule encoding a catalytic domain of a fungal exo-cellobiohydrolase, a DNA molecule encoding a catalytic domain of an endoglucanase, and a terminator. 
     
     
         16 . The vector according to  claim 15  further comprising a selectable marker. 
     
     
         17 . The vector according to  claim 15  further comprising a linker located 3′ of the exo-cellobiohydrolase (CBH) catalytic domain and 5′ of the EG catalytic domain. 
     
     
         18 . The vector according to  claim 15  further comprising a kexin site. 
     
     
         19 . The vector according to  claim 15  wherein the catalytic domain of the endoglucanase is derived from a bacterial endoglucanase. 
     
     
         20 . A fungal host cell transformed with a heterologous exo-endo cellulase fusion construct according to  claim 1 . 
     
     
         21 . A fungal host cell transformed with a vector according to  claim 15 . 
     
     
         22 . A recombinant fungal cell comprising the heterologous exo-endo cellulase fusion construct according to  claim 1 . 
     
     
         23 . A recombinant fungal cell comprising a vector according to  claim 15 . 
     
     
         24 . The recombinant fungal cell according to  claim 22  wherein the fungal host cell is a  Trichoderma  host cell. 
     
     
         25 . The recombinant fungal cell according to  claim 22  wherein the fungal host cell is a strain of  T. reesei.    
     
     
         26 . The recombinant fungal cell according to  claim 22  wherein at least one gene selected from the group consisting of the cbh1, cbh2, egl1 and eg/2 has been deleted from the fungal cells. 
     
     
         27 . An isolated cellulase fusion protein having cellulolytic activity which comprises an exo-cellobiohydrolase catalytic domain and an endoglucanase catalytic domain. 
     
     
         28 . The isolated cellulase fusion protein according to  claim 27  wherein the exo-cellobiohydrolase is a CBH1. 
     
     
         29 . The isolated cellulase fusion protein according to  claim 27  wherein the catalytic domain of the endoglucanase is derived from a bacterial cell. 
     
     
         30 . The isolated cellulase fusion protein according to  claim 29  wherein bacterial cell is a strain of  Acidothermus cellulolyticus.    
     
     
         31 . A cellulolytic composition comprising the isolated cellulase fusion protein according to  claim 29 . 
     
     
         32 . A method of producing an enzyme having cellulolytic activity comprising, a) stably transforming a filamentous fungal host cell with a heterologous exo-endo cellulase fusion construct according to  claim 1 ; b) cultivating the transformed fungal host cell under conditions suitable for said fungal host cell to produce an enzyme having cellulolytic activity; and c) recovering said enzyme. 
     
     
         33 . The method according to  claim 32  wherein the filamentous fungal host cell is a  Trichoderma  cell. 
     
     
         34 . The method according to  claim 32  wherein the filamentous fungal host cell is a  T. reesei  host cell. 
     
     
         35 . The method according to  claim 32  wherein the exo-cellobiohydrolase is a CBH1 and the endoglucanase is selected from the group consisting of an  Acidothermus cellulolyticus  endoglucanase and a  Thermobifida fusca  endoglucanase. 
     
     
         36 . The method according to  claim 32  wherein the recovered enzyme is selected from the group consisting of a cellulase fusion protein, components of the cellulase fusion protein, and a combination of the cellulase fusion protein and the components thereof. 
     
     
         37 . The method according to  claim 32  wherein the recovered enzyme(s) is purified. 
     
     
         38 . A  Trichoderma  host cell which expresses a cellulase fusion protein, wherein said fusion protein comprises a catalytic domain of an exo-cellobiohydrolase and a catalytic domain of an endoglucanase. 
     
     
         39 . The  Trichoderma  host cell according to  claim 38  wherein the host cell is a  T. reesei  cell. 
     
     
         40 . The  Trichoderma  host cell according to  claim 38  wherein the exo-cellobiohydrolase is a CBH1 and the endoglucanase is an  Acidothermus cellulolyticus  endoglucanase. 
     
     
         41 . The  Trichoderma  host cell according to  claim 38  wherein the endoglucanase is either an  Acidothermus cellulolyticus  E1 or GH74 endoglucanase. 
     
     
         42 . The  Trichoderma  host cell according to  claim 38  wherein at least one gene selected from the group consisting of the cbh1, cbh2, egl1 and egl2 has been deleted from the host cell. 
     
     
         43 . A fungal cellulase composition comprising a cellulase fusion protein or components thereof, wherein the fusion protein or components thereof is the product of a recombinant  Trichoderma  spp. according to  claim 38 . 
     
     
         44 . A fungal cellulase composition according to  claim 43  wherein the cellulase fusion protein is a CBH1- Acidothermus cellulolyticus  E1 fusion protein and the components are the cleaved products, CBH1 and  Acidothermus  E1.