Comparative proteomics and network analysis of the secretome of thermotolerant Ascomycetes for lignin degradation.

Lignin is one of the major bottlenecks for lignocellulosic biomass conversion into biofuels and value-added bioproducts by its recalcitrance. Isolates of (Aspergillus caespitosus) show very similar absolute ligninolytic activity, but the molecular basis for the superior ligninolytic performance of different isolates remains largely elusive. Here we investigated proteomic differences between four previously characterized high- and low-performing isolates of A. caespitosus (S1, S3, S6 and S10). Proteomic analysis of secretome pairs S3 vs. S1 and S6 vs. S10 was determined comparatively by two-dimensional gel electrophoresis (2D-GE) followed by identification of the differentially expressed proteins using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Secretome proteins identified from experiments were analysed in silico for signal peptides, protein-targeting signatures and transmembrane regions using SignalP, WoLF PSORT and Phobius, respectively; predicted protein-protein interaction networks were assessed using STRING. The proteomic analysis identified a unique secretome signature for the high-performing S3 isolate with particularly strong upregulation of candidate protein Spot ID 18 (A0A3D8RZ57). The absence of transmembrane helices is predicted by the Phobius analysis, consistent with our prediction that it resides extracellularly. Then, the molecular modelling and common molecular docking were carried out (as described in detail below) to examine how the representative candidate protein interacts with xylobiose and subsequently provides further structural support for its prioritization as a lignocellulose-associated candidate, known to display interactions with xylan. Network analysis also placed Spot ID 18 at or near the centre of the predicted network of interactions, indicating a high degree of connectivity, with mounting evidence for its involvement in an orchestrated process targeting extracellular degradation functions. On the other hand, the low-performing isolates were shown to have less connected protein interaction networks. In summary, these approaches, combined, represent a systems-level framework for candidate protein identification and prioritization of lignocellulose-degradation-associated proteins in thermotolerant Ascomycetes.