Fig. 1

Modification of the his1 locus during strain generations. A In the uridine auxotrophic recipient strain T. reesei QM6a Δpyr4, the his1 gene (blue arrow) is located in close vicinity to two other genes (grey arrows; TRIREDRAFT_67534 is a predicted protein kinase, TRIREDRAFT_23028 is a hypothetical Ca2  +  permeable channel). After transformation of the plasmid pCD-Δhis1, homologous recombination may occur at the two flanks (orange and yellow boxes) resulting in the replacement of the his1 promoter and a part of the coding sequence with the A. fumigatus pyrG marker (green arrow), which restores uridine prototrophy. This yields the strain T. reesei QM6a Δhis1 (pyrG  +). B Due to the direct repeat of a part of the 5′flank (dark orange box) in front of the 3′flank (yellow box) in the strain T. reesei QM6a Δhis1 (pyrG  +) an internal homologous recombination may occur spontaneously, which leads to the loss of the pyrG gene. This results in uridine auxotrophy and the generation of the double-auxotrophic strain T. reesei QM6a Δpyr4 Δhis1. C Transformation of the plasmid pCD-ReHis-eyfp into the strain T. reesei QM6a Δpyr4 Δhis1 may lead to a homologous recombination at the 5′ and 3′flanks (orange and yellow boxes). As the plasmid contains the previously deleted his1 promoter and partial coding region, the native his1 locus is restored and additionally an EYFP expression cassette integrated upstream of the his1 promoter, yielding the strain QM6a Δpyr4 eyfp(his1)