Unravelling the Potential of Fungal Division of Labour in the Laccase Producer <em>Coriolopsis trogii</em> MUT3379

The white-rot fungus Coriolopsis trogii MUT3379 produces laccase Lac3379-1 in high yields due to the previous implementation of a robust fermentation process. Throughout the extended use of this strain, we observed the occurrence of substrate-specific and transient alternative guaiacol and ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) oxidizing enzymes. Since we could not produce these enzymes in significant amounts using 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 C. trogii MUT3379. A cost-effective and efficient protocol for protoplast preparation was developed using the enzymatic cocktail VinoTaste Pro by Novozymes. A total of 100 protoplast-derived clones were selected and screened to produce laccases and/or other oxidative enzymes. A variable spectrum of oxidative activity levels, including both high and low producers, was revealed. Notably, a subset of clones exhibited different guaiacol/ABTS positive enzymatic patterns. These findings suggest that it is possible to separate different lineages from the mycelium of C. trogii MUT337 producing a different pattern of oxidative enzymes, unravelling the potential of fungal division of labour to discover novel metabolic traits that otherwise remain cryptic. These data hold outstanding significance for accessing and producing novel oxidative enzymes from native fungal populations.