Flammulina filiformis is one of the widely produced edible fungi worldwide. It is rich in γ-aminobutyric acid (GABA), a non-protein amino acid with important physiological functions in humans. To investigate the functions of key genes in the GABA metabolic pathway of F. filiformis, we isolated the monokaryon Fv-HL23-1 from the factory-cultivated F. filiformis strain Fv-HL23 and then sequenced and assembled the genome using the PacBio Sequel and Illumina NovaSeq sequencing platforms. The results showed that the genome comprised 140 scaffolds with a total length of 40.96 Mb, a GC content of 49.62%, an N50 of 917,125 bp, and 14,256 protein-coding genes. Phylogenetic analysis based on the whole genome revealed a close evolutionary relationship of Fv-HL23-1 with Armillaria mellea, Lentinula edodes, and Schizophyllum commune. A total of 589 carbohydrate-active enzymes were identified in the genome of Fv-HL23-1, suggesting its strong lignocellulose degradation ability, and 108 CYP450 gene family members were identified, suggesting important functions such as resistance to stress, secondary metabolite synthesis, and growth and development. The F. filiformis proteins glutamate decarboxylase 1 (Ff-GAD1) and glutamate decarboxylase 2 (Ff-GAD2), which may be responsible for GABA synthesis, were identified by protein alignment. Molecular docking analysis showed that Ff-GAD2 may have better catalytic activity than Ff-GAD1. To verify the function of Ff-gad2, its heterologous expression in the mycelia of the mononuclear Hypsizigus marmoreus was analyzed. Compared with wild type, the GABA content of mycelia was increased by 85.40–283.90%, the growth rate was increased by 9.39 ± 2.35%, and the fresh weight was increased by 18.44 ± 7.57%. Ff-GAD2 may play a catalytic role in GABA synthesis. In addition, the expression of the full-length Ff-gad2 gene was increased by 7.96 ± 1.39 times compared with the exon expression level in H. marmoreus mycelia, suggesting that the intron may contribute to the heterologous expression of Ff-GAD2. Based on whole-genome sequencing, we analyzed the enzyme system related to the important life activities of F. filiformis, focusing on the function of Ff-GAD, a key enzyme in the GABA synthesis pathway. The results lay a foundation for elucidating the GABA metabolism pathway of edible fungi and developing targeted breeding strategies for GABA-producing edible fungi.

γ-Aminobutyric (GABA) acid is a nutrient and signaling molecule existing in many plants, participating in the regulation of metabolism and various physiological activities. Two strains of Hypsizygus marmoreus (a white variety and a brown variety) were investigated to study the impact of exogenous GABA on mycelial growth and the response to stress. Mycelial growth, microscopic morphology, antioxidant profile, and gad2 expression in H. marmoreu were investigated under salt, dehydration, or cold stress. The results indicated that 5 mM GABA stimulated mycelial growth under standard cultivation conditions, whereas GABA addition over 10 mM hindered the growth. Under salt, dehydration, or cold stress, treatment with 5 mM GABA significantly enhanced the mycelial growth rate and density of both H. marmoreus strains by promoting front hyphae branching. Meanwhile, the activities of key antioxidant enzymes such as peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were enhanced by GABA, thereby augmenting the defensive network against abiotic stress. Additionally, gad2 expression and GABA concentration were increased under abiotic stresses as a resistance regulation response. The exogenous addition of GABA strengthened the upregulation of gad2 expression and GABA production. These findings indicated that exogenously adding low concentrations of GABA effectively enhanced the mycelial growth and antioxidant profile of H. marmoreus, thereby improving its resistance against stresses.

Mitochondria regulate nuclear genomes and their own genetic material, primarily to provide energy in eukaryotes. Currently, high-throughput sequencing technologies are being used to resolve the mitochondrial genomes of various edible fungi. However, the application of pan-genomes for the analysis of edible mushroom mitochondrial genomes remains unexplored. In this study, we conducted a comparative mitochondrial genome analysis of 31 Hypsizygus marmoreus strains (four newly sequenced monotypes and 27 public datasets), ranging from 98,284 to 111,087 bp. This variation was determined to be primarily driven by dynamic changes in non-coding regions, particularly intronic polymorphisms in the cox1 gene. Further, transfer RNA (tRNA) secondary structures exhibited atypical globular and elongated conformations alongside copy number variations. Additionally, codon usage showed a pronounced A/T bias, whereas core respiratory chain genes demonstrated an evolutionary pattern of strong purifying selection. Furthermore, the 31 mitochondrial genomes of H. marmoreus were found to harbor eight gene rearrangement patterns and five genetic clusters, and the pan-genome analysis (220,364 bp, 217 nodes) captured abundant single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and structural variations. This study provides breeding-relevant genetic markers and a genomic framework for H. marmoreus germplasm classification, genetic improvements, and the molecular breeding of stress-resilient varieties.

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