Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System

Copper (Cu) homeostasis has not been well-documented in filamentous fungi, especially extremophiles. Acidophilic fungus Acidomyces richmondensis MEY-1 has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA has been shown to be involved in this process. The ArAceA deletion mutant (?ArAceA) exhibits specific growth defects at Cu concentrations of = 10 mM. To gain genomic insight into the Cu tolerance mechanism and the role of ArAceA in Cu tolerance, we treated the ?ArAceA mutant and WT strains with or without 15 mM CuCl2 for 6 h. Transcriptional profiling analysis revealed that ?ArAceA mutant is transcriptionally more sensitive to Cu than the wild-type strain. Our findings provide insights into the molecular basis of Cu tolerance in acidophilic filamentous fungi. Overall design: Arthroconidia of WT and ?ArAceA strain were inoculated into 100 mL of liquid MMC and grown at 30°C and 200 rpm for 4 days. The mycelium was washed twice with sterilized water and then transferred to 100 mL of MMC, with or without addition of 15 mM CuCl2, for 6 h before RNA extraction. Triplicate cultures were sampled in each condition.