Proteomics Insights into the Biomass Hydrolysis Potentials of a Hypercellulolytic Fungus Penicillium funiculosum

Abstract

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

Comparative insights into the saccharification potentials of a relatively unexplored but robust Penicillium funiculosum glycoside hydrolase 7 cellobiohydrolase

“…Penicillium funiculosum (NCIM 1228) previously identified and characterized as a hypercellulolytic fungus [5] was maintained on potato dextrose agar and cultivated in a cellulase-inducing medium containing soya peptone (24 g/L), KH 2 PO 4 (5.9 g/L), (NH4) 2 SO 4 (3.12 g/L), CaCl 2 ·2H 2 O (0.05 g/L), yeast extract (0.05 g/L), wheat bran (24 g/L) and Avicel (21.4 g/L); the final pH was adjusted to 5.5. The cellulase inducing medium in Erlenmeyer flasks was inoculated with four plugs (1 mm diameter) from the edge of the actively growing fungi, respectively.…”

“…The activities of the purified enzyme towards cellobiohydrolase substrates—microcrystalline cellulose (Avicel PH-101), and chromogenic substrates— p -nitrophenyl-β- d -cellobioside ( p NPC) and from p -nitrophenyl-β- d -lactopyranoside ( p NPL) all obtained from Sigma, USA were measured as described earlier [5]. All experiments were conducted in 96 wells deep well plates.…”

“…All pipetting mix were performed on Tecan Freedom Evo-2 150 Liquid Handler Automated workstation (Tecan Group Ltd., Switzerland). On completion of the dispensing program, the plates were sealed with adhesive PCR Plate Seals (Genetix, India) to prevent evaporation and biomass hydrolysis was carried out as previously reported [5]. …”

“…They are mainly found in eukaryotes, of which reports of discoveries in filamentous fungi predominates, and are among the most common cellulolytic enzymes in secretomes of biomass-degrading fungi produced under cellulase-inducing conditions [3–5]. They act from the reducing ends of cellulose chains, latching onto the cellulose substrates and processively release cellobiose until they run into obstructions or are inactivated [6–8].…”

“…Our previous work on the strain identified it as a hypercellulolytic fungus. We also discovered that it has only one gene coding for GH7 cellobiohydrolase (PfCBH1) and that the enzyme is possibly the most important protein in cellulose-hydrolyzing secretome based on its abundance and distribution [5]. However, the properties of the enzyme had not been previously explored or reported.…”