Protein production in filamentous fungal cells in the absence of inducing substrates

Abstract

Certain embodiments of the disclosure are directed to variant filamentous fungal cells, compositions thereof and methods thereof for increased production of one or more proteins of interest. More particularly, in certain embodiments, the disclosure is directed to variant filamentous fungal (host) cells derived from parental filamentous fungal cells, wherein the variant host cells comprise a genetic modification which enables the expression/production of a protein of interest (POI) in the absence of inducing substrate. In certain embodiments, a variant fungal host cell of the disclosure comprises a genetic modification which increases the expression of a variant activator of cellulase expression 3 (ace3) gene encoding an Ace3 protein referred to herein as Ace3-L.

1 - 16 . (canceled) 
     
     
         17 . A method for producing a lignocellulosic degrading enzyme in a  Trichoderma  sp. fungal cell in the absence of an inducing substrate comprising:
 introducing into the fungal cell a polynucleotide construct comprising an upstream (5′) promoter sequence operably linked to a downstream (3′) nucleic acid encoding an Ace3 protein comprising at least 95% sequence identity to SEQ ID NO: 6 and “Lys-Ala-Ser-Asp” as the last four C-terminal amino acids, and   fermenting the cell under conditions suitable for fungal cell growth and protein production, wherein such conditions do not include an inducing substrate.   
     
     
         18 . The method of  claim 17 , wherein the polynucleotide construct further comprises a downstream (3′) native ace3 terminator sequence operably linked to the nucleic acid encoding the Ace3 protein. 
     
     
         19 . The method of  claim 17 , wherein the polynucleotide construct is integrated into the fungal cell genome. 
     
     
         20 . The method of  claim 17 , wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The method of  claim 17 , wherein the encoded Ace3 protein comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. 
     
     
         24 - 57 . (canceled) 
     
     
         58 . The method of  claim 17 , wherein the lignocellulose degrading enzyme is selected from the group consisting of cellulase enzymes, hemi-cellulase enzymes, or a combination thereof. 
     
     
         59 . The method of  claim 17 , further comprising an introduced expression cassette encoding a protein of interest.