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

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.