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

Flammulina filiformis is one of the widely produced edible fungi worldwide. It is rich in &gamma:-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&ndash:283.90%, the growth rate was increased by 9.39 &plusmn: 2.35%, and the fresh weight was increased by 18.44 &plusmn: 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 &plusmn: 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.