Patrick Jara

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA+)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica

SummaryEndothiapepsin 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 wildtype 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.

Self-cloning in filamentous fungi: Application to the construction of endothiapepsin overproducers in Cryphonectria parasitica

The filamentous ascomycete fungus Cryphonectria parasitica naturally secretes endothiapepsin, an aspartic proteinase. It is cultured on a commercial scale as a source of the milk-clotting enzyme for cheese making. Our objective was to increase enzyme production of an industrial C. parasitica strain by a new technique of self-cloning; it consisted in the screening for transformants producing higher levels of endothiapepsin and having integrated only the DNA fragment of interest. Such genetically improved strains that are devoid of any foreign genes should be more readily acceptable for the production of food-grade enzymes.