Characterization of the Ku70 homologue HdfA deletion in Penicillium chrysogenum. Penicillium chrysogenum

Targeting an engineered DNA fragment to a specific site in chromosomes in order to disrupt, overexpress or modify the nucleotide sequence of a gene requires homologous recombination repair mechanism. This DNA repair mechanism is not predominant in fungi, resulting in extremely low targeting efficiency. To increase this efficiency, it is becoming common practice to disable the non homologous end joining (NHEJ) pathway that causes random integration, by deleting homologous gene to human KU70 and KU80 which encode proteins functioning in the NHEJ pathway. These genes have been successfully deleted in several organisms, including the yeast Kluyveromyces lactis and the fungi Neurospora Crassa and several Aspergilli species. In this study we investigated the behavior of high penicillinG-producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues, HdfA or HdfB, had been deleted. Targeting efficiency in these mutant strains was significantly increased relative to the reference strain. Both physiological and transcriptome data of chemostat cultivations of the hdfA deletion strain and the reference strain showed minimal differences. However, in a direct competition experiment to assess global strain fitness, the reference strain had a clear advantage over the deletion strain. The full characterization of these recombinant host strains is an essential step to guide the future construction of a whole genome knock-out mutant collection. Overall design: Penicillium chrysogenum is a filamentous fungus that is used to industrially produce the beta-lactam antibiotics. After decades of classical strain improvement, the genome sequence has been deciphered and annotated. The combination of the P. chrysogenum genome sequence and a strain exhibiting increased targeting efficiency would paved the way to the construction of deletion or expression libraries that would be a tremendous help in understanding the nature of the productive capabilities of this organism. This study reports the effect of the deletions of the Ku70 and Ku80 homologues, HdfA and HdfB respectively, in P. chrysogenum. A detailed analysis of the physiology, transcriptome and phenotype was performed to assess the use of these strains as platform for functional genomics study.