Extracellular enzyme activities on day 10.

Ganoderic acids (GAs) are bioactive triterpenoids produced by Ganoderma species with demonstrated anticancer properties. While the yield of GAs in Ganoderma tsugae is typically low, heat stress has been shown to enhance its production. This study employed atmospheric and room temperature plasma (ARTP) mutagenesis to develop thermotolerant G. tsugae mutants for high-temperature cultivation at 35°C. From 59 mutants generated, strain Ganoderma tsugae 9 (GT9) demonstrated superior thermotolerance, showing 51.48% increased mycelial growth rate and 76.03% higher biomass compared to wild-type (WT) at 35°C (one-way ANOVA with Dunnett’s test, p hmgr, sqs, se, ls) and heat shock protein genes (hsp17.4, hsp22, hsp70, hsp90). After 10-day cultivation at 35°C, GT9 produced 1.01-fold more GAs than the WT at 35°C and 22.64% more than the WT at 25°C (two-way ANOVA with Tukey’s test, p G. tsugae mutants enable efficient high-temperature fermentation for enhanced GAs production. This strategy provides significant advantages for industrial-scale application while elucidating the physiological and molecular mechanisms underlying improved GAs biosynthesis under heat stress.