Development of a CRISPR/Cas9-Based Marker Recycling System in Trametes versicolor via pyrG Complementation

White-rot fungi possess a unique ability to produce diverse lignin-degrading enzymes and degrade complex polymers. These fungi potentially offer significant potential for various industrial applications, such as bioremediation and the production of bioactive compounds. Accordingly, research has been increasingly focused on exploring the metabolic pathways and biological mechanisms. Among these fungi, Trametes versicolor has been reported in recent studies to efficiently degrade industrial waste. However, research progress in mushroom species has been significantly limited compared to that in other organisms due to the absence of antibiotic resistance markers, the low efficiency of homology-directed repair (HDR), and the lack of a universal transformation protocol. To address these limitations, we developed a marker recycling system by generating a pyrG deletion strain using CRISPR/Cas9. First, we generated pyrG deletion strain capable of surviving on 5-fluoroorotic acid (5-FOA) media. By integrating a wild-type pyrG cassette into the target gene of ΔpyrG strain, successful selection was confirmed through the restoration of growth on uracil- and uridine- deficient media. Subsequent PCR analysis verified the precise deletion of the target gene. This strategy provides an efficient approach for marker utilization in T. versicolor and will contribute to the advancement of molecular genetic tools for exploring the diverse functional genomics.