Efficient gene targeting in Aspergillus niger using a transiently disrupted ku70 gene

Mark Arentshorst, Arthur F.J. Ram and Vera Meyer

Author address: 

Leiden University, IBL, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, The Netherlands. [email protected]

Abstract: 

Homologous recombination frequencies in filamentous fungi, including Aspergillus niger, are low. Several reports over the last years have shown that mutants defective in the Non-Homologous-End Joining (NHEJ) pathway display increased homologous integration efficiencies up to 80 to 100%. Recently, we described that deletion of the A. niger kusA gene, encoding the orthologue of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency (1). However, deletion of kusA also causes increased sensitivity of A. niger towards UV and X-ray and the consequences of loss of kusA in relation to DNA repair and genome stability are currently unknown. To avoid any potential side effects of a kusA loss-of-function mutation on growth and viability of A. niger, we transiently disrupted kusA. W e made use of the counter selectable amdS marker, flanked by 300 bp direct repeats of the kusA gene. Disruption of the kusA gene resulted in similar homologous recombination frequencies compared to the deltakusA strain. After completion of the gene targeting approach, we re-established an intact kusA copy using counter selection on fluoroacetamide, proven by sequencing of the kusA locus. In A. niger, which lacks an sexual reproduction cycle, the transient disruption system is especially important as the NHEJ pathway cannot be restored by performing a sexual cross. References: 1. Meyer, V., Arentshorst, M., El-Ghezal, A., Drews, A.C., Kooistra, R., van den Hondel, C.A.M.J.J., Ram, A.F.J. Journal of Biotechnology, 128 (4), 770-5. 2007.
2010

abstract No: 

22.

Full conference title: 

7th International Aspergillus Meeting
    • Asperfest 7 (2010)