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

A method for introducing an alteration into a target nucleic acid sequence contained in a cell, comprising:

• (a) obtaining mutant cells of a parent cell by introducing into the parent cell a nucleic acid construct comprising a nitrate reductase selectable marker gene, flanked by nucleotide sequence repeats to form a marker cassette, wherein the repeats consist of a nucleotide sequence immediately 5′ or a nucleotide sequence immediately 3′ of the target DNA;
• and further wherein
• when the flanking nucleotide sequence repeats consist of a nucleotide sequence 5′ of the target DNA, the nucleic acid construct further comprises, 3′ of, and contiguous to, the marker cassette, an additional nucleotide sequence consisting of a nucleotide sequence 3′ of the target DNA sequence;
• and wherein
• when the flanking nucleotide sequence repeats consist of a nucleotide sequence 3′ of the target DNA, the nucleic acid construct further comprises, 5′ of, and contiguous to, the marker cassette, an additional nucleotide sequence consisting of a nucleotide sequence 5′ of the target DNA sequence;
• (b) selecting mutant cells from step (a) by selecting for the presence of the nitrate reductase gene;
• (c) cultivating the selected mutant cells under conditions effecting deletion of the nitrate reductase gene by recombination between the flanking nucleotide sequence repeats;
• and
• (d) selecting a mutant cell with an alteration in the target nucleic acid sequence in which the nitrate reductase gene has been deleted from the mutant cell.