Production of secreted proteins by filamentous fungi

Abstract

The present invention relates to a method to improve the secretion of a protein of interest by a filamentous fungal cell comprising inducing a phenotype in the cell selected from the group consisting of a lowered ERAD, an elevated UPR that does not induce an elevated ERAD, wherein ERAD preferably is lowered. The invention further relates to the filamentous fungal cell comprising the phenotype described above. The invention also relates to polynucleotides and polypeptides whose expression can be modulated in the filamentous fungal cell to obtain the above-described phenotype.

1 . Method to improve the secretion of a protein of interest by a filamentous fungal cell comprising inducing a phenotype in the cell selected from the group consisting of:
 (i) a lowered ERAD,   (ii) an elevated UPR that does not induce an elevated ERAD,   (iii) an elevated UPR that does not induce an elevated ERAD, wherein ERAD is lowered   
     
     
         2 . Method to improve the secretion of a protein of interest by a filamentous fungal cell, wherein the expression of a DNA sequence is modulated, said DNA sequence selected from the group consisting of SEQ ID NO's: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, or a homologue thereof. 
     
     
         3 . Method according to  claim 2 , wherein the expression of at least one DNA sequence selected from the group consisting of SEQ ID NO's: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, or a homologue thereof, is modulated, preferably up regulated. 
     
     
         4 . Method according to  claim 2  or  3 , wherein the expression of at least one DNA sequence selected from the group consisting of SEQ ID NO's: 43, 46, 49, 52, 55, 58, or a homologue thereof, is modulated, preferably down regulated. 
     
     
         5 . Method according to  claims 2  to  4 , characterized in that the expression level of at least one of the DNA sequences having the following SEQ ID NO or homologues thereof given below or a combination of at least one taken from each subgroup a), b) c), d), or e) given below, or a combination thereof is up regulated:
 a) 4, 25, 34, 40, 
 b) 25, 
 c) 10, 13, 22, 25, 28, 31, 31, 
 d) 25, 
 e) 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 
 and/or wherein the expression level of at least one of the DNA sequences selected from the group consisting of SEQ ID NO's: 43, 46, 49, 52, 55, 58, or homologues thereof, or a combination thereof is down regulated. 
 
     
     
         6 . Method according to  claims 1  to  5 , wherein expression of the native sec61 gene is preferably impaired, said method further comprising production in the filamentous fungal cell of:
 (i) a polypeptide according to SEQ ID NO: 63, and/or 
 (ii) a polypeptide according to SEQ ID NO: 63, wherein the amino acid at position 376 is replaced by phenylalanine, tyrosine or histidine. 
 
     
     
         7 . Method according to any one of  claims 2  to  6 , characterized in that the expression level of a DNA sequence which is down regulated is lower in the obtained filamentous fungus than the expression level of the corresponding DNA sequence in the parental filamentous fungus the filamentous fungus originates from, preferably three times lower, more preferably four times lower, most preferably more than four times lower and even most preferably not detectable using northern, or western blotting or array technique. 
     
     
         8 . Method according to any one of  claims 2  to  7 , characterized in that the expression level of a DNA sequence which is up regulated is higher in the obtained filamentous fungus than the expression level of the corresponding DNA sequence in the parental filamentous fungus the filamentous fungus originates from, preferably three times higher, more preferably four times higher and most preferably more than four times higher using northern, or western blotting or array technique. 
     
     
         9 . Method according to any one of  claims 1  to  8 , wherein the filamentous fungus is selected from the group consisting of the genera  Aspergillus, Trichoderma  and  Penicillium.    
     
     
         10 . Method according to  claim 9 , characterized in that the  Aspergillus  is selected from the species  Aspergillus niger, Aspergillus oryzae, Aspergillus  sojae and the  Trichoderma  is selected from the species  Trichoderma reesei.    
     
     
         11 . Polynucleotide comprising a DNA sequence selected from the group consisting of SEQ ID NO's: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, or a homologue thereof, or a degenerated DNA sequence obtainable there from. 
     
     
         12 . Polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO's: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, or a homologue thereof. 
     
     
         13 . Filamentous fungus displaying a modulated expression of at least one DNA sequence selected from the group consisting of SEQ ID NO's: SEQ ID NO's: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, or a homologue thereof. 
     
     
         14 . Filamentous fungus, wherein the expression of at least one DNA sequence selected from the group consisting of SEQ ID NO's: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, or a homologue thereof, is modulated, preferably up regulated. 
     
     
         15 . Filamentous fungus, wherein the expression of at least one DNA sequence selected from the group consisting of SEQ ID NO's: 43, 46, 49, 52, 55, 58, or a homologue thereof, is modulated, preferably down regulated. 
     
     
         16 . Filamentous fungus, wherein the expression level of at least one of the DNA sequences having the following SEQ ID NO or homologues thereof given below or a combination of at least one taken from each subgroup a), b) c), d), or e) given below, or a combination thereof is up regulated:
 a) 4, 25, 34, 40,   b) 25,   c) 10, 13, 22, 25, 28, 31, 31,   d) 25,   e) 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40,   and/or wherein the expression level of at least one of the DNA sequences selected from the group consisting of SEQ ID NO's: 43, 46, 49, 52, 55, 58, or homologues thereof, or a combination thereof is down regulated.   
     
     
         17 . Filamentous fungus according to  claims 14  to  18 , wherein expression of the native sec61 gene is preferably impaired, said filamentous fungus further synthesising intracellularly:
 (i) a sec61 polypeptide according to SEQ ID NO: 63, and/or 
 (ii) a sec61 polypeptide according to SEQ ID NO: 63, wherein the amino acid at position 376 is replaced by phenylalanine, tyrosine or histidine. 
 
     
     
         18 . Filamentous fungus according to  claims 13  to  17 , transformed with the polynucleotide of  claim 12 . 
     
     
         19 . Filamentous fungus according to  claim 13  or  18 , wherein the filamentous fungus further comprises a DNA sequence encoding a protein of interest. 
     
     
         20 . Filamentous fungus according to  claim 19 , wherein the DNA sequence encoding a protein of interest is operably linked to a promoter and to a secretion signal. 
     
     
         21 . Method for the production of a protein of interest comprising culturing the filamentous fungus of  claim 19  or  20  under conditions conducive to expression of the protein and optionally recovering the produced protein.