Unravelling the Potential of Fungal Division of Labour in the Laccase Producer Coriolopsis trogii MUT3379 Through Protoplast Formation and Regeneration

The white-rot fungus <i>Coriolopsis trogii</i> MUT3379 produces Lac3379-1 laccase at high yields due to the previous development of a robust fermentation process. Throughout the extended use of this strain, we observed the occurrence of substrate-specific guaiacol and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) oxidizing enzymes other than Lac3379-1 Since we did not succeed in producing these enzymes in significant amounts by conventional strain selection and fermentation tools, we developed an approach based on protoplast preparation and regeneration to isolate stable producers of these alternative oxidative enzymes from the complex multinucleate mycelium of <i>C. trogii</i> MUT3379. A cost-effective and efficient protocol for protoplast preparation was developed by using the enzymatic cocktail VinoTaste Pro by Novozymes. A total of 100 protoplast-derived clones were selected and screened to produce laccases and other oxidative enzymes. A variable spectrum of oxidative activity levels, including both high and low producers, was revealed. Notably, a subset of clones exhibited diverse guaiacol/ABTS positive enzymatic patterns. These findings suggest that it is possible to isolate different lineages from the mycelium of <i>C. trogii</i> MUT337, each producing a distinct pattern of oxidative enzymes. This highlights the potential of protoplast-mediated genome separation to uncover novel metabolic traits that would otherwise remain cryptic. These data hold outstanding significance for accessing and producing novel oxidative enzymes from native fungal populations.