Process optimization for protoplast preparation from Grifola frondosa and development of a high-yield mutant with...

Grifola frondosa is a highly valued medicinal mushroom with diverse bioactive properties; however, its low mycelial biomass remains a major bottleneck affecting the efficiency of liquid fermentation. To address this issue, protoplast-mediated mutagenesis was employed in this study to breed a high-performance G. frondosa mutant with superior fermentation traits. Following optimization using intelligent strategies, the highest yield of protoplasts was achieved (6.573 × 10⁶ protoplasts/g fresh weight) under suitable isolation parameters (fungal age of 7.9 days, lywallzyme concentration 2.1%, enzymatic hydrolysis time 5.2 hours, enzymatic temperature of 30 °C, and mannitol concentration of 0.6 mol/L). Subsequently, DES-induced protoplast mutagenesis yielded a stable G. frondosa mutant (D24) showing 62.78% greater mycelial biomass and 111.11% higher protein yield relative to the parental strain. Furthermore, fermentation kinetics profiling and phenotypic observation suggested that the mutant D24 exhibited a shortened lag phase, enhanced hyphal dispersion, and denser mitochondrial distribution compared with the original strain. Additionally, genome resequencing identified 57,216 single nucleotide polymorphisms (SNPs) in D24 relative to the original strain, including 4,068 non-synonymous SNPs. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment indicated that the mutated genes were potentially involved in amino acid synthesis and metabolism, lipid metabolism, and others. These candidate mutations may be associated with the increased mycelial biomass and protein content. Collectively, this study has developed a novel high-yielding mutant of G. frondosa, which may serve as a promising material for future pharmaceutical and biotechnological applications.