Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same

Abstract

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3′ terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

What is claimed is:  
     
         1 . A vector comprising a DNA sequence capable of expressing a heterologous polypeptide in a filamentous fungus and of causing secretion of said heterologous polypeptide from said filamentous fungus.  
     
     
         2 . The vector of  claim 1  wherein said secreted heterologous polypeptide is biochemically active.  
     
     
         3 . The vector of  claim 1  wherein said heterologous polypeptide is an enzyme.  
     
     
         4 . The vector of  claim 3  wherein said enzyme is selected from the group consisting of chymosin, prochymosin, preprochymosin,  A. niger  glucoamylase,  Humicola grisea  glucoamylase and  Mucor mehei  carboxyl protease.  
     
     
         5 . A DNA sequence encoding a heterologous polypeptide, said DNA sequence being capable of being expressed in a filamentous fungus and of causing secretion of said heterologous polypeptide.  
     
     
         6 . The DNA sequence of  claim 5  wherein said secreted heterologous polypeptide is biochemically active.  
     
     
         7 . The DNA sequence of  claim 5  wherein said heterologous polypeptide is an enzyme.  
     
     
         8 . The DNA sequence of  claim 7  wherein said enzyme is selected from the group consisting of chymosin, prochymosin, preprochymosin,  A. niger  glucoamylase,  Humicola grisea  glucoamylase and  Mucor mehei  carboxyl protease.  
     
     
         9 . A vector for transforming a filamentous fungus comprising a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence operably linked thereto, said signal sequence being functional in a secretory system in said filamentous fungus.  
     
     
         10 . The vector of  claim 9  wherein said signal sequence is native to said heterologous polypeptide.  
     
     
         11 . The vector of  claim 10  wherein said native signal sequence and said heterologous sequence are selected from the group consisting of DNA sequences encoding bovine preprochymosin,  Mucor meihei  preprocarboxyl protease and  A. niger  preproglucoamylase.  
     
     
         12 . The vector of  claim 9  wherein said signal sequence is foreign to said heterologous polypeptide.  
     
     
         13 . The vector of  claim 12  wherein said foreign signal sequence is selected from the group consisting of DNA sequences encoding the signal sequence of bovine preprochymosin,  A. niger  glucoamylase and  Mucor miehei  carboxyl protease.  
     
     
         14 . The vector of  claim 9  further comprising a DNA sequence encoding a promoter sequence functionally recognized by said filamentous fungus including transcription and translation control sequences operably linked to said DNA sequence encoding said signal sequence.  
     
     
         15 . The vector of  claim 14  wherein said DNA sequence encoding said promoter sequence is selected from the group consisting of DNA sequences encoding the promoter sequence of  A. niger  glucoamylase and  Mucor miehei  carboxyl protease.  
     
     
         16 . The vector of  claim 9  further comprising DNA sequences encoding a functional polyadenylation sequence operably linked to said DNA sequence encoding said heterologous polypeptide.  
     
     
         17 . The vector of  claim 16  wherein said polyadenylation sequence is selected from the group consisting of DNA sequences encoding the polyadenylation sequence of  A. niger  glucoamylase or  Mucor meihei  carboxyl protease.  
     
     
         18 . The vector of  claim 9  further comprising a DNA sequence encoding a selection characteristic expressible in said filamentous fungus.  
     
     
         19 . The vector of  claim 18  wherein said selection characteristic is selected from the group consisting of DNA sequences encoding  N. crassa  pyr4,  A. nidulans  acetamidase,  A. nidulans  argB and  A. nidulans  trpC.  
     
     
         20 . The vector of  claim 9  further comprising a DNA sequence capable of increasing the transformation efficiency of said vector into said filamentous fungus.  
     
     
         21 . The vector of  claim 20  wherein said DNA sequence for increasing fungal transformation efficiency is ANS-1.  
     
     
         22 . The vector of  claim 9  wherein said secreted heterologous polypeptide is an enzyme.  
     
     
         23 . The vector of  claim 22  wherein said enzyme is selected from the group consisting of chymosin, prochymosin, preprochymosin,  A. niger  glucoamylase,  Humicola grisea  glucoamylase and  Mucor mehei  carboxyl protease.  
     
     
         24 . The vector of  claim 9  wherein said secreted heterologous polypeptide is a mammallian polypeptide.  
     
     
         25 . The vector of  claim 9  wherein said secreted heterologous polypeptide is biochemically active.  
     
     
         26 . A filamentous fungus containing a vector selected from the group consisting of the vectors of claims  1  through  25 .  
     
     
         27 . A filamentous fungus capable of expressing and secreting a heterologous polypeptide.  
     
     
         28 . The filamentous fungus of  claim 27  wherein said heterologous polypeptide is integrated into the geneome of said filamentous fungus.  
     
     
         29 . The filamentous fungus of  claim 27  wherein said filamentous fungus is selected from the group consisting of Aspergillus species, Trichoderma species and Mucor species.  
     
     
         30 . The filamentous fungus of  claim 27  wherein said filamentous fungus is  A. nidulans  or  A. awamori.    
     
     
         31 . The filamentous fungus of  claim 27  wherein said filamentous fungus is  Trichoderma reesei.    
     
     
         32 . The filamentous fungus of  claim 27  wherein said heterologous polypeptide is an enzyme selected from the group consisting of chymosin, prochymosin, preprochymosin,  A. niger  glucoamylase,  Humicola grisea  glucoamylase and  Mucor mehei  carboxyl protease.  
     
     
         33 . The filamentous fungus of  claim 27  wherein said secreted heterologous polypeptide is a mammalian polypeptide.  
     
     
         34 . The filamentous fungus of  claim 27  wherein said secreted heterologous polypeptide is biochemically active.  
     
     
         35 . A process for making a heterologous polypeptide comprising: 
 transforming a filamentous fungus with a vector containing DNA sequences capable of expressing a heterologous polypeptide and of causing secretion of said heterologous polypeptide from said filamentous fungus, and    expressing and secreting said heterologous polypeptide.    
     
     
         36 . The process of  claim 35  wherein said expressing and secreting is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements.  
     
     
         37 . The process of  claim 35  further comprising the step of isolating said secreted heterologous polypeptide.  
     
     
         38 . The process of  claim 35  wherein said vector containing DNA sequences capable of expressing a heterologous polypeptide and of causing secretion of said heterologous polypeptide is selected from the group consisting of the vectors of claims  1  through  25 .  
     
     
         39 . The process of  claim 35  wherein said secreted heterologous polypeptide is an enzyme selected from the group consisting of chymosin, prochymosin, preprochymosin,  A. niger  glucoamylase,  Humicola grisea  glucoamylase and  Mucor mehei  carboxyl protease.  
     
     
         40 . The process of  claim 35  wherein said secreted heterologous polypeptide is a mammalian polypeptide.  
     
     
         41 . The process of  claim 35  wherein said secreted heterologous polypeptide is biochemically active.