Deletion of ligD significantly improves gene targeting frequency in the lignocellulolytic filamentous fungus Penicillium oxalicum

Abstract

To improve the gene targeting frequency (GTF) in the lignocellulolytic filamentous fungus Penicillium oxalicum HP7-1, the non-homologous end-joining (NHEJ) gene ligD was deleted. The obtained PoligD deletion mutant ΔPoligD showed no apparent defect in cellulase production, growth rate, and sensitivity towards osmotic stress and mutagen ethyl methanesulphonate (EMS), while increased sensitivity to high concentrations of methyl methanesulfonate (MMS). Deletion of PoligD gene resulted in significantly increased GTFs at three different loci in P. oxalicum, which are even higher than those in Poku70 deletion mutant. The GTF in ΔPoligD at PoargB (reached 97 %) and PoagaA (reached 90 %) loci increased 5.1- and 1.2-fold compared with that in wild-type strain (WT), while at the Podpp4 locus GTF was up to 27 % in ΔPoligD but close to 0 % in WT, with 0.5 kb homologous flanking regions. Furthermore, the argB and agaA nutritional selection in P. oxalicum was demonstrated and the PoargB and PoagaA genes could be used as selective markers in this fungus. Thus, the PoligD deletion mutant can be an important tool for the functional analysis of genes in P. oxalicum.