Whole-Genome Sequence Analysis of Flammulina filiformis and Functional Validation of Gad, a Key Gene for γ-Aminobutyric Acid Synthesis

<i>Flammulina filiformis</i> 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 <i>F. filiformis</i>, we isolated the monokaryon <i>Fv-HL23-1</i> from the factory-cultivated <i>F. filiformis</i> strain <i>Fv-HL23</i> 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 <i>Fv-HL23-1</i> with <i>Armillaria mellea</i>, <i>Lentinula edodes</i>, and <i>Schizophyllum commune</i>. A total of 589 carbohydrate-active enzymes were identified in the genome of <i>Fv-HL23-1</i>, 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 <i>F. filiformis</i> 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 <i>Ff-gad2</i>, its heterologous expression in the mycelia of the mononuclear <i>Hypsizigus marmoreus</i> 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 <i>Ff-gad2</i> gene was increased by 7.96 ± 1.39 times compared with the exon expression level in <i>H. marmoreus</i> 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 <i>F</i>. <i>filiformis</i>, 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.