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Auxotrophic mutants of  Aspergillus  can be isolated in the presence of counter-selective compounds, but the process is laborious. We developed a method to enable reversible impairment of the  ku80  gene ( Aaku80 ) in the imperfect fungus  Aspergillus aculeatus .  Aaku80  was replaced with a selection marker, orotidine 5’-phosphate decarboxylase ( pyrG ), followed by excision of  pyrG  between direct repeats (DR) to yield the  Aaku80  deletion mutant (MR12). The gene-targeting efficiency at the ornithine carbamoyltransferase ( argB ) locus was drastically elevated from 3% to 96% in MR12. The frequency of marker recycling depended on DR length. One uridine auxotroph was obtained from 3.3 × 10 5 , 1.4 × 10 5 , and 9.2 × 10 3  conidia from strains harboring 20-, 98-, and 495-bp DRs, respectively. Because these strains maintained the short DRs after 5 d of cultivation, we investigated whether  Aaku80  function was disrupted by  pyrG  insertion with the 20-bp DR and restored after excision of  pyrG . The  Aaku80  disruption mutant (coku80) was bred by inserting  pyrG  sandwiched between 20-bp DRs into the second intron of  Aaku80 , followed by excision of  pyrG  between the DRs to yield the coku80rec strain. Analyses of homologous recombination frequency and methyl methanesulfonate sensitivity demonstrated that  Aaku80  function was disrupted in coku80 but restored in coku80rec. Furthermore,  pyrG  was maintained in coku80 at least for ten generations. These data indicated that reversible impairment of  ku80  in  A .  aculeatus  is useful for functional genomics in cases where genetic segregation is not feasible.

Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus aculeatus