Transcriptomic analysis of two ascomycete fungi on the utilization of tobacco biomass and identification of key pectinases in polysaccharides deconstruction

Tobacco waste, the lignocellulosic biomass generated during tobacco processing, is a potentially sustainable resource for biotechnological applications. The microbial conversion of tobacco waste relies on enzymes secreted by microorganisms, with filamentous fungi playing a crucial role in lignocellulose degradation. However, the transcriptomic responses and molecular basis of fungi in response to tobacco-derived biomass have not yet been extensively explored. In this study, we assessed the main organic components, including cellulose, pectin, starch, and lignin, in four different tobacco-derived materials and revealed distinct variations in their contents. The industrial fungus Myceliophthora thermophila and the model fungus Neurospora crassa were selected for growth assays on tobacco waste and employed for comparative transcriptomic profiling. Transcriptomic analysis of M. thermophila and N. crassa revealed significant upregulation of 87 and 60 carbohydrate-active enzymes and 27 and 11 transcription regulators, respectively, under tobacco induction. Notably, three pectinases, MtPly-1, MtPly-2, and NcPly-2, exhibited markedly elevated expression levels across tobacco induction conditions. These pectinases were overexpressed in M. thermophila using a viral 2A peptide strategy, and their biochemical properties were characterized. The recombinant enzymes demonstrated activity and stability within the range of pH 6.0–11.0, with MtPly-1 and MtPly-2 exhibiting enhanced thermostability. Furthermore, the addition of either MtPly-1 or MtPly-2 significantly improved the saccharification efficiency of tobacco biomass, surpassing the activity of M. thermophila cellulase alone. These findings suggest the potential of MtPly-1 and MtPly-2 as thermostable pectinases for efficient degradation of tobacco biomass, with promising applications in biomass saccharification and tobacco fermentation processes in industrial biotechnology. • M. thermophila and N. crassa are potential candidates for degrading tobacco biomass. • MtPly-1, MtPly-2, and Ncply-2 were most strongly upregulated under tobacco biomass. • Important roles for pectinases MtPly-1 or MtPly-2 in the biomass saccharification.