Phanerochaete chrysosporium is a model white-rot-fungus with exceptional lignolytic activity, but its genetic manipulation has remained a major challenge due to low transformation efficiencies. Here, we report the development of an optimized Agrobacterium-mediated transformation (AMT) protocol optimized for this species. A CRISPR-Cas9 compatible binary vector was modified by replacing the mCherry cassette with a strong sGFP reporter under the constitutive 35S promoter, enabling reliable identification of transformants. Using mycelial disks as starting material and applying a sandwich selection method, we achieved stable integration of T-DNA carrying the hygromycin resistance and sGFP genes. Transformation efficiency was influenced by co-cultivation parameters, with low-temperature incubation (23°C) and the addition of acetosyringone significantly improving recovery of resistant colonies. Stable expression of sGFP was observed in transformed mycelia, confirming the reproducibility of the system. Although CRISPR-Cas9-mediated gene editing was not demonstrated in this study, our results provide proof-of-concept that a Cas9-based vector can be stably maintained in P. chrysosporium and drive heterologous gene expression. This work represents the first step toward establishing a functional genome editing platform in this ligninolytic fungus. By expanding the molecular toolbox for P. chrysosporium, the optimized transformation strategy lays the foundation for metabolic engineering aimed at lignin valorization, bioplastics, biofuels, and high-value biochemical production within a sustainable circular bioeconomy framework.
