Development of an efficient PEG-Mediated protoplast transformation system for the medicinal fungus Ophiocordyceps xuefengensis

Ophiocordyceps xuefengensis is a newly identified medicinal fungus with significant pharmacological and economic value, but its genetic manipulation has been impeded by the lack of an efficient transformation system. Here, we established the first stable and highly efficient polyethylene glycol (PEG)-mediated protoplast transformation platform for O. xuefengensis using hygromycin B as a selectable marker. Through systematic optimization of critical parameters such as enzyme composition, enzyme concentration, mycelial age, and digestion conditions, we developed an optimized protocol for protoplast preparation. A high protoplast yield of 9.42x107 CFU/mL was achieved using 4-day-old mycelia with 1.5% lywallzyme 1 and 1.5% snailase digested at 34{whitebullet}C with shaking at 130 rpm for 3.5 h. PY medium containing 0.6 M mannitol significantly enhanced protoplast regeneration. Stable plasmid integration and robust hygR gene expression were confirmed through PCR detection and sustained antibiotic resistance over four successive generations. Furthermore, a controllable expression system was established by using the endogenous promoter that drove the stable expression of the glycoside hydrolase gene cbhI. The result of enzymatic assay confirmed the functional production of CBHI enzyme, demonstrating the credibility of this system. This work provides a reliable genetic toolbox for functional genomics studies, targeted gene manipulation, and strain engineering in O. xuefengensis, facilitating fundamental research and sustainable utilization of this valuable medicinal species. IMPORTANCEThe conservation and sustainable utilization of medicinal fungi represents a critical challenge in biotechnology and natural product research. Ophiocordyceps xuefengensis, a newly identified species with significant pharmacological potential, exemplifies this challenge: its wild resources are diminishing while artificial cultivation remains restricted by limited understanding of its fundamental biology. The lack of genetic tools has impeded progress in elucidating its biosynthetic pathways, regulating fruiting body development, and enhancing metabolite production. We established a highly efficient PEG-mediated transformation system that directly addresses this technological gap. In addition, we demonstrated that this transformation system can support heterologous gene expression using endogenous promoters. This platform enables reliable genetic manipulation in O. xuefengensis, permitting functional genomics studies and targeted bioactive compounds synthesis.