Proteomic insights into Pholiota nameko mycelial response to wood-based substrates

Pholiota microspora (Berk.) Sacc., commonly known as "nameko," is an edible mushroom widely cultivated in East Asia for both culinary and medicinal purposes. This study aimed to investigate how substrate composition influences the expression of lignocellulolytic enzymes in Pholiota nameko using label-free quantitative proteomics. The fungus was cultivated on four substrates: (1) a standard commercial mix (S) of oak sawdust, millet, rye, and wheat bran (24:8:3:3 w/w); (2) a minimally supplemented willow-based substrate (S1) with the same ratio; (3) a mixture of willow chips and cottonseed hulls (W + C) in a 3:1 ratio; and (4) willow chips alone (W). All substrates were incubated at a suboptimal temperature of 15 °C to simulate low-energy cultivation conditions and assess enzymatic responses under thermal constraint. A total of 1783 proteins were identified, with 165 showing statistically significant differential expression across substrates. Functional annotation revealed diverse enzyme groups, including oxidoreductases (e.g., manganese peroxidases, peroxygenases, tyrosinases) and hydrolases (e.g., endoglucanases, exoglucanases), which exhibited substrate-dependent regulation. Notably, oxidative enzymes were predominantly upregulated in the standard substrate, S while cellulolytic enzymes showed enhanced expression in substrates W and W + C. Several uncharacterized proteins were also identified, highlighting the need for future functional studies. These results underscore P. nameko's adaptive enzymatic responses to lignocellulosic substrates under suboptimal, low-temperature conditions. This work advances understanding of its lignocellulolytic enzyme system, with implications for energy-efficient mushroom cultivation and industrial enzyme production.