Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants

Inactivating the non-homologous end-joining (NHEJ) pathway is a well established method to increase gene targeting (GT) efficiencies in filamentous fungi. In this study we have compared the effect of inactivating the NHEJ genes ku70 or lig4 on CT in the industrial penicillin producer Penicillium chrysogenum. Deletion of both genes resulted in strongly increased CT efficiencies at three different loci but not higher than 70%, implying that other, yet uncharacterized, recombination pathways are still active causing a part of the DNA to be integrated via non-homologous recombination. To further increase the CT efficiency we applied the bi-partite approach, in which the DNA fragment for integration was split in two non-functional overlapping parts that via homologous recombination in vivo can form a functional selection marker. The combined NHEJ mutant and bi-partite approach further increased CT frequencies up to similar to 90%, which will enable the efficient high throughput engineering of the P. chrysogenum genome. We expect that this combined approach will function with similar high efficiencies in other filamentous fungi. (C) 2010 Elsevier Inc. All rights reserved.