Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica

Endothiapepsin is an aspartic protease secreted by Cryphonectria parasitica. It has a milk-clotting activity and is used in the cheese industry. The eapA gene encoding endothiapepsin has been cloned and sequenced. An open reading frame of 419 codons, which encodes a precursor differing from mature endothiapepsin by the presence of an 89 aa residue prepro-sequence, was found. The eapA gene is interrupted by three introns. C. parasitica mutant strains deficient in the production of endothiapepsin (eapA-) were constructed using a gene-replacement strategy. Two nonsense mutations were introduced at the beginning of the coding sequence by PCR-induced mutagenesis. The mutated DNA fragment was introduced in C. parasitica by co-transformation with a benomyl-resistant (benR) selection plasmid. Transformants which have the eapA- phenotype were obtained. Protein analysis confirmed that they secreted no detectable amount of endothiapepsin. No ectopic integration of the mutated eapA gene occurred in the eapA- transformants. Moreover, after one conidiation step, eapA- transformants yielded benomyl-sensitive (benS) segregants which were analyzed by Southern blotting experiments. The results revealed no difference with the wild-type strain, suggesting that the eapA-, benS segregants differed from the non-transformed strain only by the presence of the two nonsense mutations in the eapA locus.