Copper-responsive lcc3 gene confers laccase (LCC3) hyperproduction and oxidative stress resistance in the ligninolytic fungus Dichomitus squalens

AIMS: This study aimed to elucidate how copper ions (Cu2+) control laccase production in Dichomitus squalens, to identify the Cu2+-responsive laccase gene(s), and to clarify their physiological roles. METHODS AND RESULTS: The research employed various experimental approaches including enzyme activity assays, transcriptome sequencing, proteomics, RT-qPCR, RNA interference, and ROS detection. Laccase activity and biomass of D. squalens were quantified after exposure to 0-3.0 mM Cu2+, showing that 2.0 mM Cu2+ elevated total laccase activity without inhibiting mycelial growth. Transcriptome sequencing and RT-qPCR revealed that the expression of laccase gene lcc3 was specifically up-regulated by 299.8-fold. Quantitative proteomics revealed that the abundance of LCC3 protein, the predominant extracellular laccase isoform, rose 44.8-fold upon copper supplementation, underscoring its pivotal contribution to overall laccase activity. In addition, RNA interference of lcc3 gene markedly reduced Cu2+-stimulated laccase activity even under copper induction, confirming its indispensable role. Finally, the total reactive oxygen species (ROS) in Cu2+-treated hyphae were lower than in untreated controls, linking lcc3 gene induction to cellular redox homeostasis. CONCLUSIONS: In this study, by integrating transcriptomics, proteomics and functional genetics, we elucidated that Cu2+ specifically induces the expression of specific laccase genes to enhance laccase activity in D. squalens. lcc3 is the sole laccase gene that is massively and specifically induced by physiological Cu2+ concentrations. We further clarified the function of its encoded protein: LCC3 protein not only enhances extracellular laccase activity but also confers Cu2+ tolerance via ROS scavenging and Cu2+ chelation. These findings uncover a unique copper-responsive regulon and establish the lcc3 gene as a promising target for metabolic engineering aimed at high-yield laccase production and robust fungal performance under metal stress.