Systematic identification of CAZymes and transcription factors in the hypercellulolytic fungus Penicillium funiculosum NCIM1228 involved in lignocellulosic biomass degradation.

BACKGROUND: Penicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes. Since transcription factors (TFs) are the key players in regulating the expression of enzymes, this study aimed first to identify the complete repertoire of Carbohydrate Active Enzymes (CAZymes) and TFs coded in its genome. The regulation of CAZymes was then analysed by studying the expression pattern of these CAZymes and TFs in different carbon substrates-Avicel (cellulosic substrate), wheat bran (WB; hemicellulosic substrate), Avicel + wheat bran, pre-treated wheat straw (a potential substrate for lignocellulosic ethanol), and glucose (control). RESULTS: The P. funiculosum NCIM1228 genome was sequenced, and 10,739 genes were identified in its genome. These genes included a total of 298 CAZymes and 451 TF coding genes. A distinct expression pattern of the CAZymes was observed in different carbon substrates tested. Core cellulose hydrolyzing enzymes were highly expressed in the presence of Avicel, while pre-treated wheat straw and Avicel + wheat bran induced a mixture of CAZymes because of their heterogeneous nature. Wheat bran mainly induced hemicellulases, and the least number of CAZymes were expressed in glucose. TFs also exhibited distinct expression patterns in each of the carbon substrates. Though most of these TFs have not been functionally characterized before, homologs of NosA, Fcr1, and ATF21, which have been known to be involved in fruiting body development, protein secretion and stress response, were identified. CONCLUSIONS: Overall, the P. funiculosum NCIM1228 genome was sequenced, and the CAZymes and TFs present in its genome were annotated. The expression of the CAZymes and TFs in response to various polymeric sugars present in the lignocellulosic biomass was identified. This work thus provides a comprehensive mapping of transcription factors (TFs) involved in regulating the production of biomass hydrolyzing enzymes.

Quantitative multiplexed profiling of Penicillium funiculosum secretome grown on polymeric cellulase inducers and glucose.

Filamentous fungi respond to the need to secure utilisable carbon from their growth milieu by secreting unique extracellular proteins depending upon the types of polymeric substrates. We have here profiled the variations in the secretome pattern of a non-model hypercellulolytic fungus - Penicillium funiculosum, grown in minimal media containing four different polymeric cellulase inducers, i.e., Avicel, wheat bran, ammonium-pretreated wheat straw and Avicel & wheat bran, and glucose over its early and late log phases of growth. Of the 137 secreted proteins validated at 1% FDR, we identified the quantified proteins in three clusters as early, persistently or lately expressed. The type of carbon substrate present in the culture media significantly affected the levels of cellulolytic enzymes expression by the fungus. The top abundant proteins quantified in the secretome for Avicel and wheat bran were cellobiohydrolaseI [GH7-CBM1], cellobiohydrolaseII [GH6-CBM1], ß-glucosidase [GH3], arabinofuranosidase [GH51] and ß-xylosidase [GH3], with bicupin being highest in case of wheat straw. Our results further suggested that the fungus secreted the extracellular proteins in waves, such that the initial responders act to hydrolyse the composite substrates in the culture environment before the second wave of proteins which tend to be more tailored to the specific substrate in the cultivating media. BIOLOGICAL SIGNIFICANCE: In this article, we have comprehensively examined the dynamics of the secretome of a non-model hypercellulolytic fungus produced in response to model and composite cellulase inducers. Our study has provided additional insights into how the fungus enzyme machinery responds to the presence of different polymeric cellulase inducers over the two different growth phases (early growth and late growth phase). The comprehensive typing and quantification of the different proteins present in the secretomes of the cellulolytic fungal strains in response to diverse nutrient sources hold many prospects in understanding the fungus unique enzyme machinery and dynamics for the downstream biotechnological applications.

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

BACKGROUND: GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete-Penicillium funiculosum (NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse-Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus. RESULTS: There were marginal differences observed in the stability of both enzymes, with P. funiculosum (PfCBH1) showing an optimal thermal midpoint (Tm) of 68 °C at pH 4.4 as against an optimal Tm of 65 °C at pH 4.7 for T. reesei (TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (Km), an 18-fold higher turnover rate (kcat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (Ki) than TrCBH1 on p-nitrophenyl-ß-d-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2-G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads-EXDXXE motifs-were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted. CONCLUSION: To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete-P. funiculosum NCIM1228, against the backdrop of the same enzyme from the industrial workhorse-T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 from T. reesei in industrial cellulase cocktails.

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

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.

Effect of carbon and nitrogen sources on polygalacturonase production by Trichoderma viride (BITRS-1001) isolated from tar sand in Ondo State, Nigeria

The effects of the various carbon and nitrogen substrates on the growth and polygalacturonase activity of Trichoderma viride (BITRS-1001) isolated from the tar sand deposit in Gbelejuloda-Irele Ondo State, Nigeria were investigated in submerged cultivation at 30 °C ± 2 °C. The commercial carbon and nitrogen substrates included sucrose, fructose, starch, maltose, lactose and peptone, sodium nitrate, urea and casein respectively. All the carbon substrates used supported the growth of T. viride (0.566 to 0.156 g/50 mL of culture medium) with starch supporting the highest biomass yield and sucrose the least biomass yield. Maximum polygalacturonase activity of 3033 U/mL was recorded in maltose medium. Maximum biomass yield on the nitrogen sources was observed in the organic nitrogen namely peptone and casein with values not significantly different from each other at p ≤ 0.05. In the determination of the crude enzyme activity on the nitrogen sources, maximum polygalacturonase activity of 12,400 U/mL was recorded in peptone medium. Hence, a careful manipulation of these nutrient substrates could help to optimise the production of this enzyme on a large scale.