Methods For Producing Secreted Polypeptides Having Biological Activity

Abstract

The present invention relates to methods for producing a polypeptide having biological activity, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first polynucleotide encoding the polypeptide operably linked to a second polynucleotide encoding a variant signal peptide or a variant prepropeptide; and (b) isolating the secreted polypeptide having biological activity from the cultivation medium.

1 - 31 . (canceled) 
     
     
         32 . A genetically engineered and isolated polynucleotide, which encodes a prepropeptide variant consisting of amino acids 1 to 17 of SEQ ID NO: 2 without amino acids “SPIRR” wherein the encoded prepropeptide variant is directly fused in frame to the amino terminus of a mature polypeptide of interest which is encoded by said polynucleotide. 
     
     
         33 . The polynucleotide of  claim 32 , wherein the nucleotide sequence encoding the mature polypeptide encodes a heterologous polypeptide. 
     
     
         34 . The polynucleotide of  claim 32 , wherein the nucleotide sequence encoding the mature polypeptide encodes a lipase. 
     
     
         35 . A nucleic acid construct comprising the polynucleotide of  claim 32 . 
     
     
         36 . The nucleic acid construct of  claim 35 , wherein the polynucleotide encoding the prepropeptide variant is operably linked to a second polynucleotide encoding a polypeptide, wherein the 3′ end of the prepropeptide variant polynucleotide is immediately upstream of the start codon of the second polynucleotide. 
     
     
         37 . A recombinant host cell transformed with the nucleic acid construct of  claim 36 . 
     
     
         38 . The recombinant host cell of  claim 37 , wherein the host cell is a yeast or filamentous fungal host cell. 
     
     
         39 . The recombinant host cell of  claim 38 , wherein the yeast host cell is a  Saccharomyces  host cell. 
     
     
         40 . The recombinant host cell of  claim 39 , wherein the  Saccharomyces  host cell is a  Saccharomyces cerevisiae  host cell. 
     
     
         41 . The recombinant host cell of  claim 38 , wherein the filamentous fungal host cell is an  Aspergillus  host cell. 
     
     
         42 . The recombinant host cell of  claim 41 , wherein the  Aspergillus  host cell is an  Aspergillus oryzae  host cell. 
     
     
         43 . A method for producing a mature polypeptide, comprising: (a) cultivating the recombinant host cell of  claim 37  in a medium conducive for the production of the mature polypeptide, wherein said host cell produces said mature polypeptide at a higher yield compared to an otherwise identical host cell that expresses amino acids 1 to 22 of SEQ ID NO:
 2 directly fused in frame to the amino terminus of the encoded mature polypeptide. 
 
     
     
         44 . The method of  claim 43 , wherein the recombinant host cell is a yeast or filamentous fungal host cell. 
     
     
         45 . The method of  claim 44 , wherein the yeast host cell is a  Saccharomyces  host cell. 
     
     
         46 . The method of  claim 45 , wherein the  Saccharomyces  host cell is a  Saccharomyces cerevisiae  host cell. 
     
     
         47 . The method of  claim 44 , wherein the filamentous fungal host cell is an  Aspergillus  host cell. 
     
     
         48 . The method of  claim 47 , wherein the  Aspergillus  host cell is an  Aspergillus oryzae  host cell. 
     
     
         49 . The method of  claim 43 , wherein the recombinant host cell produces at least 25% more mature polypeptide relative to an otherwise identical host cell that expresses amino acids 1 to 17 of SEQ ID NO: 2 without amino acids “SPIRR” directly fused in frame to the amino terminus of the encoded mature polypeptide. 
     
     
         50 . The method of  claim 43 , wherein the nucleotide sequence encoding the mature polypeptide encodes a heterologous polypeptide. 
     
     
         51 . The method of  claim 43 , wherein the nucleotide sequence encoding the mature polypeptide encodes a lipase.