Methods for producing heterologous polypeptides in trichothecene-deficient filamentous fungal mutant cells

Abstract

The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a mutant of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes involved in the production of a trichothecene and (ii) the mutant produces less of the trichothecene than the parent filamentous fungal cell when cultured under the same conditions; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells. The present invention also relates to isolated trichodiene synthases and isolated nucleic acid sequences encoding the trichodiene synthases. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the trichodiene synthases. The present invention further relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.

1 . A method for obtaining a mutant  Trichoderma  cell, comprising:
 (a) introducing into an endogenous gene of the genome of a parent  Trichoderma  cell, a nucleic acid construct comprising a nucleic acid sequence of the endogenous gene inactivated with a nitrate reductase gene to produce a mutant  Trichoderma  cell comprising an endogenous gene incapable of expressing the endogenous gene product, wherein the endogenous gene is one or more genes selected from the group consisting of a tri3 gene, a tri4 gene, a tri5 gene, a tri6 gene, a trill gene, a tri12 gene, and a tril01 gene;   (b) selecting the resulting mutant  Trichoderma  cell from step (a) for the presence of the nitrate reductase gene and loss of production of the gene product;   (c) introducing into the mutant  Trichoderma  cell from step (b) a second nucleic acid construct comprising a second inactivated nucleic acid of the endogenous gene sequence comprising 5′ and 3′ regions of the inactivated nucleic acid sequence of the endogenous gene, but lacking the nitrate reductase gene, wherein the second nucleic acid construct incorporates into the genome of the mutant  Trichoderma  cell replacing the inactivated nucleic acid sequence and is incapable of expressing the endogenous gene product; and   (d) selecting a mutant  Trichoderma  cell from step (c) in the absence of the nitrate reductase gene.   
     
     
         2 . The method of  claim 1 , wherein the endogenous gene is a tri3 gene. 
     
     
         3 . The method of  claim 1 , wherein the endogenous gene is a tri4 gene. 
     
     
         4 . The method of  claim 1 , wherein the endogenous gene is a tri5 gene. 
     
     
         5 . The method of  claim 1 , wherein the endogenous gene is a tri6 gene. 
     
     
         6 . The method of  claim 1 , wherein the endogenous nucleic acid sequence is a tri11 gene. 
     
     
         7 . The method of  claim 1 , wherein the endogenous gene is a tri12 gene. 
     
     
         8 . The method of  claim 1 , wherein the endogenous gene is a tri101 gene. 
     
     
         9 . The method of  claim 1 , wherein the parent  Trichoderma  cell is a  Trichoderma harzianum  cell. 
     
     
         10 . The method of  claim 1 , wherein the parent  Trichoderma  cell is a  Trichoderma koningii  cell. 
     
     
         11 . The method of  claim 1 , wherein the parent  Trichoderma  cell is a  Trichoderma longibrachiatum  cell. 
     
     
         12 . The method of  claim 1 , wherein the parent  Trichoderma  cell is a  Trichoderma reesei  cell. 
     
     
         13 . The method of  claim 1 , wherein the parent  Trichoderma  cell is a  Trichoderma viride  cell.