Thermostable trichoderma cellulase

Abstract

Described are compositions and methods relating to the thermostable fungal cellulase enzyme, EGV, and Trichoderma host cells having a modification comprising or consisting essentially of disruption or deletion of nucleotide(s) for expression of this cellulose, whereby EGV expression is prevented.

1 . A  Trichoderma  host cell comprising:
 a modification wherein the modification substantially reduces or prevents production of a thermostable endoglucananse V (EGV) by the  Trichoderma  host cell, and   an exogenous nucleic acid molecule of interest encoding a polypeptide of interest operably linked to a promoter whereby at least one endogenous polypeptide and the polypeptide of interest is each expressed in vivo by the  Trichoderma  host cell.   
     
     
         2 . The  Trichoderma  host cell of  claim 1  wherein the modification comprises one or more deletion or disruption in nucleotides involved in expression of EGV. 
     
     
         3 . The  Trichoderma  host cell of  claim 2  wherein the disruption or deletion comprises deletion or disruption of the gene encoding or the coding region for or the promoter or regulatory elements for expression of EGV. 
     
     
         4 . The  Trichoderma  host cell of  claim 3 , wherein the deletion or disruption comprises deletion or disruption of egl5. 
     
     
         5 . The  Trichoderma  host cell of  claim 4  wherein the deletion or disruption comprises egl5 disruption. 
     
     
         6 . The  Trichoderma  host cell of  claim 4  wherein the deletion or disruption comprises egl5 deletion. 
     
     
         7 . The  Trichoderma  host cell of  claim 6 , wherein egl5 is deleted by homologous recombination. 
     
     
         8 . The  Trichoderma  host cell of  claim 1 , wherein the endogenous polypeptide comprises a cellulase, a hemi-cellulase, a protease, or combinations thereof. 
     
     
         9 . The  Trichoderma  host cell of  claim 8 , wherein the thermolabile enzyme comprises a cellulase or a hemi-cellulase. 
     
     
         10 . The  Trichoderma  host cell of  claim 1  wherein the  Trichoderma  is  T. reesei.    
     
     
         11 . The  Trichoderma  host cell of  claim 1 , wherein the polypeptide of interest is hydrophobin II. 
     
     
         12 . The  Trichoderma  host cell of  claim 10 , wherein the polypeptide of interest is hydrophobin II. 
     
     
         13 . The  Trichoderma  host cell of  claim 1 , wherein the polypeptide of interest is thermostable. 
     
     
         14 . The  Trichoderma  host cell of  claim 1 , wherein the polypeptide of interest is heat sensitive or thermolabile. 
     
     
         15 . A method for making the polypeptide of interest in the modified  Trichoderma  host cell of  claim 1 , wherein the polypeptide of interest is thermostabile, comprising:
 incubating the modified host cell in a medium suitable for the modified host cell to produce the thermostable polypeptide of interest and the endogenous polypeptide, whereby each of the endogenous polypeptide and the thermostable polypeptide of interest each is expressed by the modified host cell; and   subjecting the thermostable polypeptide of interest and the endogenous polypeptide to an elevated temperature,   wherein the elevated temperature is sufficient to substantially inactivate the endogenous polypeptide, but insufficient to inactivate the thermostable polypeptide of interest and EGV if it were present;   whereby the thermostable polypeptide of interest is produced in active or functional form substantially in the absence of activity from the endogenous polypeptide.   
     
     
         16 . The method of  claim 15  further comprising isolating a protein mixture from the modified  Trichoderma  host cell prior to the step of subjecting the polypeptide of interest and the endogenous polypeptide to the elevated temperature. 
     
     
         17 . The method of  claim 15  wherein the modified host cell expresses an exo-cellobiohydrolase, an endoglucanase, a β-glucosidase, or combinations thereof. 
     
     
         18 . The method of  claim 15 , wherein the thermostable polypeptide of interest is reversibly heat-denaturable. 
     
     
         19 . The method of  claim 15 , wherein the elevated temperature is a temperature of about 90° C. or more. 
     
     
         20 . The method of  claim 15 , wherein exposure to the elevated temperature is for a time of about 5 minutes or more. 
     
     
         21 . The method of  claim 15 , wherein exposure to the elevated temperature is for a time of about 60 minutes or more. 
     
     
         22 . A method for controlling activity of the polypeptide of interest expressed by the modified  Trichoderma  host cell of  claim 1 , wherein the polypeptide of interest is heat sensitive or thermolabile, said method comprising subjecting the heat sensitive or thermolabile polypeptide of interest and the endogenous polypeptide to an elevated temperature,
 wherein the elevated temperature is sufficient to inactivate the endogenous polypeptide and the heat sensitive or thermolabile polypeptide.   
     
     
         23 . The method of  claim 22 , wherein the endogenous polypeptide is a cellulase. 
     
     
         24 . The method of  claim 22 , further comprising, prior to subjecting the heat sensitive or thermolabile polypeptide of interest to the elevated temperature, contacting the polypeptide of interest with a compound upon which the polypeptide of interest acts 
     
     
         25 . The method of  claim 23 , further comprising, prior to subjecting the heat sensitive or thermolabile polypeptide of interest to the elevated temperature, contacting the polypeptide of interest with one or more compounds upon which the cellulose and the polypeptide of interest act. 
     
     
         26 . A fermentation broth composition obtained or obtainable from filamentous fungus host cell and comprising hydrophobin II, said fermentation broth being substantially free of cellulase and/or mannanase activity, and wherein the hydrophobin II is produced in a modified  Trichoderma  host cell according to  claim 11 . 
     
     
         27 . A fermentation broth composition obtained or obtainable from the modified  Trichomderma  of  claim 1 . 
     
     
         28 . A method for controlling activity of the polypeptide of interest in the fermentation broth of  claim 27 , wherein the polypeptide of interest is heat sensitive or thermolabile, said method comprising subjecting the fermentation broth to an elevated temperature sufficient to inactivate the endogenous polypeptide and the heat-sensitive or thermolabile polypeptide. 
     
     
         29 . The method of  claim 28 , wherein the endogenous polypeptide is a cellulase. 
     
     
         30 . The method of  claim 28 , further comprising, prior to subjecting the fermentation broth to the elevated temperature, contacting the fermentation broth with a compound of interest upon which the polypeptide of interest acts. 
     
     
         31 . The method of  claim 29 , further comprising, prior to subjecting the fermentation broth to the elevated temperature, contacting the fermentation broth with one or more compounds upon which the celulase and the polypeptide of interest act. 
     
     
         32 . A method for controlling the activity of a cellulase present in a composition obtained from the modified  Trichoderma  host cell of  claim 1 , wherein the composition comprises at least one cellulase enzyme other than EGV, said method comprising subjecting the composition to an elevated temperature sufficient to inactivate the polypeptide of interest, and wherein following subjecting the composition to an elevated temperature the composition is substantially free of cellulase activity. 
     
     
         33 . The method of  claim 32 , wherein the polypeptide of interest is heat sensitive or thermolabile. 
     
     
         34 . The method of  claim 32 , wherein the polypeptide of interest is heat tolerant or thermostable. 
     
     
         35 . The method of  claim 32 , wherein subjecting the composition to an elevated temperature is performed in a foodstuff or beverage. 
     
     
         36 . The method of  claim 32 , wherein the elevated temperature is less than about 100 degrees C. 
     
     
         37 . The method of  claim 32 , wherein the composition obtained from the modified  Trichoderma  host cells is a hydrophobin-comprising fermentation broth.