An Efficient and Stable PEG-Mediated Transformation System for Medicinal Fungus Ophiocordyceps xuefengensis: Optimization and Functional Validation

<i>Ophiocordyceps xuefengensis</i> is an important medicinal fungus with considerable pharmaceutical and economic value. However, its industrial and scientific utilization has been severely limited by the lack of an efficient genetic transformation system, largely due to limited genomic information and wild growth. In this study, we established an efficient and stable plasmid transformation system within <i>O. xuefengensis</i> protoplasts mediated by PEG. To overcome low protoplast yield and transformation efficiency, key factors influencing protoplast preparation including enzyme composition and concentration, fungal age, and digestion conditions were systematically optimized. The optimal protocol involved digesting 4-day-old mycelia with a mixture of 1.5% lywallzyme 1 and 1.5% snailase at 34 °C and 130 rpm for 3.5 h, yielding at least 9.42 × 10⁷ CFU/mL protoplasts. Protoplast regeneration was significantly enhanced in PY medium supplemented with 0.6 M mannitol. Under these optimized conditions, a transformation efficiency of 45.5% was achieved, with stable plasmid integration confirmed over four successive generations. Furthermore, the transformation system was successfully applied to functional gene characterization by driving exogenous gene expression using the endogenous <i>gpd</i>1 promoter. This study provides a foundational platform for functional gene analysis and paves the way for further elucidation of growth and development mechanisms and metabolic engineering in <i>O. xuefengensis</i>.