Early cellular events and potential regulators of cellulase induction in Penicillium janthinellum NCIM 1366.

Cellulase production by fungi is tightly regulated in response to environmental cues, and understanding this mechanism is a key pre-requisite in the efforts to improve cellulase secretion. Based on UniProt descriptions of secreted Carbohydrate Active enZymes (CAZymes), 13 proteins of the cellulase hyper-producer Penicillium janthinellum NCIM 1366 (PJ-1366) were annotated as cellulases- 4 cellobiohydrolases (CBH), 7 endoglucanases (EG) and 2 beta glucosidases (BGL). Cellulase, xylanase, BGL and peroxidase activities were higher for cultures grown on a combination of cellulose and wheat bran, while EG was stimulated by disaccharides. Docking studies indicated that the most abundant BGL- Bgl2- has different binding sites for the substrate cellobiose and the product glucose, which helps to alleviate feedback inhibition, probably accounting for the low level of glucose tolerance exhibited. Out of the 758 transcription factors (TFs) differentially expressed on cellulose induction, 13 TFs were identified whose binding site frequencies on the promoter regions of the cellulases positively correlated with their abundance in the secretome. Further, correlation analysis of the transcriptional response of these regulators and TF-binding sites on their promoters indicated that cellulase expression is possibly preceded by up-regulation of 12 TFs and down-regulation of 16 TFs, which cumulatively regulate transcription, translation, nutrient metabolism and stress response.

Comparison of the solid-state and submerged fermentation derived secretomes of hyper-cellulolytic Penicillium janthinellum NCIM 1366 reveals the changes responsible for differences in hydrolytic performance.

Solid-state fermentation (SSF) and submerged fermentation (SmF) have often been compared for production of biomass hydrolyzing enzymes highlighting the superiority of the SSF produced enzymes, but the reasons for the performance differences are under-explored. Penicillium janthinellum NCIM 1366 culture extracts from SSF had better hydrolytic performance along with a higher initial rate of reaction. Secretome analyses of the SSF and SmF enzymes using LC/MS-MS, indicated that while the type of proteins secreted were similar in both modes, the abundance of specific beta glucosidases, lytic polysaccharide monooxygenases and hemicellulolytic enzymes were very high in SSF resulting in efficient initiation, low accumulation of cellobiose and high initial reaction rates. Key enzymes that catalyse lignocellulose breakdown under SSF and SmF are therefore different and the fungus may be speculated to have regulation mechanisms that aid differential expression under different cultivation modes.

Lignocellulose degradation by Penicillium janthinellum enzymes is influenced by its variable secretome and a unique set of feedstock characteristics.

Substrate characteristics and proteins that affect lignocellulose-hydrolysis by the hypercellulolytic fungus Penicillium janthinellum NCIM 1366 (PJ-1366) were investigated. The hydrolysis rate of PJ-1366 enzymes was very high, with upto 75 % of the reaction being completed in initial 4 h. Comparison of the hydrolytic efficiencies on differently pretreated biomass indicated that the greatest (negative) effect was imparted by lignin, suggesting that improving ligninase activity of the PJ-1366 enzymes may help to improve hydrolysis. Larger pore sizes and higher crystallinity of substrates, which favor enzyme penetration and processive hydrolysis, positively influenced hydrolysis efficiency. For alkali-pretreated substrates, 16 FPU/g of PJ-1366 cellulases released the sugar-equivalent of using 10 FPU/g of a commercial biomass hydrolyzing enzyme. By correlation analysis, 41 proteins, including 20 CAZymes were identified, whose abundance in the secretome positively correlated with the cellulase activities of the culture filtrate. These proteins may be considered as the primary drivers of FPase/CMCase/pNPGase/xylanase activity in PJ-1366.

Repurposing proteases: An in-silico analysis of the binding potential of extracellular fungal proteases with selected viral proteins.

Proteases have long been the target of many drugs, but their potential as therapeutic agents is a well-known, but under-explored area. Due to the heightened threat from new and emerging infectious agents, it is worthwhile to tap into the vast microbial protease resource to identify potential therapeutics. By docking proteases of the fungus Penicillium janthinellum NCIM 1366 with the proteins encoded by the SARS-CoV-2 virus, the enzymes that have the potential to bind with, and thereby degrade viral proteins were identified. In-silico docking analysis revealed that both fungal and commercially available proteases belonging to the A1A, M20A, S10, S8A and T1A families were able to bind the viral spike, envelope, ORF-7a and Nsp2 proteins (binding energy < -50 kJ/mol), thereby opening up the possibility of developing additional therapeutic applications for these enzymes.

Draft genome of the glucose tolerant ß-glucosidase producing rare Aspergillus unguis reveals complete cellulolytic machinery with multiple beta-glucosidase genes.

Draft genome sequence of the glucose tolerant beta glucosidase (GT-BGL) producing rare fungus Aspergillus unguis NII 08,123 was generated through Next Generation Sequencing (NGS). The genome size of the fungus was estimated to be 37.1 Mb. A total of 3116 contigs were assembled using SPades, and 15,161 proteins were predicted using AUGUSTUS 3.1. Among them, 13,850 proteins were annotated using UniProt. Distribution of CAZyme genes specifically those encoding lignocellulose degrading enzymes were analyzed and compared with those from the industrial cellulase producer Trichoderma reesei in view of the huge differences in detectable enzyme activities between the fungi, despite the ability of A. unguis to grow on lignocellulose as sole carbon source. Full length gene sequence of the inducible GT-BGL could be identified through tracing back from peptide mass fingerprint. A total of 403 CAZymes were predicted from the genome, which includes 232 glycoside hydrolases (GHs), 12 carbohydrate esterases (CEs), 109 glycosyl transferases (GTs), 15 polysaccharide lyases (PLs), and 35 genes with auxiliary activities (AAs). The high level of zinc finger motif containing transcription factors could possibly hint a tight regulation of the cellulolytic machinery, which may also explain the low cellulase activities even when a complete repertoire of cellulase degrading enzyme genes are present in the fungus.

Addressing challenges in production of cellulases for biomass hydrolysis: Targeted interventions into the genetics of cellulase producing fungi.

Lignocellulosic materials are the favoured feedstock for biorefineries due to their abundant availability and non-completion with food. Biobased technologies for refining these materials are limited mainly by the cost of biomass hydrolyzing enzymes, typically sourced from filamentous fungi. Therefore, considerable efforts have been directed at improving the quantity and quality of secreted lignocellulose degrading enzymes from fungi in order to attain overall economic viability. Process improvements and media engineering probably have reached their thresholds and further production enhancements require modifying the fungal metabolism to improve production and secretion of these enzymes. This review focusses on the types and mechanisms of action of known fungal biomass degrading enzymes, our current understanding of the genetic control exerted on their expression, and possible routes for intervention, especially on modulating catabolite repression, transcriptional regulators, signal transduction, secretion pathways etc., in order to improve enzyme productivity, activity and stability.

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30.

BACKGROUND: Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. RESULTS: Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. CONCLUSIONS: The results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.

Correction to: Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

In the original publication of the article, the affiliation of two co-authors Prajeesh Kooloth-Valappil and Meera Christopher was published incompletely. The correct affiliation of the authors should read " Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India".

Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

OBJECTIVES: Characterization of glucose tolerant beta glucosidase (GT-BGL) secreted by Aspergillus unguis NII 08123, determination of the gene and protein sequences of the enzyme and establishing its performance in blends for lignocellulose hydrolysis. RESULTS: Supplementation of A. unguis beta glucosidase (BGL) to cellulase released 1.6 times more sugar within 12 h during the hydrolysis of lignocellulosic biomass. The enzyme was determined to be similar to BGL-F from Emericella nidulans by MALDI-TOF analysis, and was found to be a GH3 family protein. Molecular Docking simulation studies showed that the enzyme has lesser affinity for glucose (- 5.7 kcal/mol) compared to its substrate cellobiose (- 7.5 kcal/mol). The residues present in the N-terminal domain are mostly involved in bond formation with both the substrate and the product, while the C-terminal domain contains the catalytic region. In-silico studies showed that its predicted structure is unlike that of previously reported BGLs, which might provide a clue to its exceptional catalytic activity. CONCLUSION: The GT-BGL from A. unguis NII 08123 was proven effective as a blend in for biomass hydrolyzing enzyme cocktails and the possible reasons for its glucose tolerance was determined through studies on its modeled structure.

A biorefinery-based approach for the production of ethanol from enzymatically hydrolysed cotton stalks.

Cotton post-harvest residue/cotton stalk (CS) - a major agro-residue in south asian countries was evaluated as a feed stock for bioethanol production. The common thermochemical pretreatment strategies based on dilute acid and alkali and different combinations of biomass hydrolyzing enzymes were evaluated for saccharification of CS biomass. A hydrolytic efficiency of 80% was achieved for alkali treated biomass using cellulase supplemented with beta glucosidase. Recycling of undigested/residual biomass and/or enzyme supported same final sugar concentration as for fresh hydrolytic experiments. Fermentation was carried out using a novel, inhibitor-resistant strain of Saccharomyces cerevisiae where 76% of theoretical maximum efficiency was attained. Material balances were derived for the entire process from biomass pre-processing to hydrolysis.

Detoxification of acidic biorefinery waste liquor for production of high value amino acid.

The current study evaluates the detoxification of acid pretreatment liquor (APL) using adsorbent (ADS 400 & ADS 800) or ion-exchange (A-27MP & A-72MP) resins and its potential for amino acid production. The APL is generated as a by-product from the pretreatment of lignocellulosic biomass and is rich monomeric sugars as well as sugar degradation products (fermentation inhibitors) such as furfural and hydroxymethyl furfural (HMF). Of the four resins compared, ADS 800 removed approximately 85% and 60% of furfural and HMF, respectively. ADS 800 could be reused for up to six cycles after regeneration without losing its adsorption properties. The study was further extended by assessing the fermentability of detoxified APL for l-lysine production using wild and mutant strains of Corynebacterium glutamicum. The detoxified APL was superior to APL for l-lysine production.

Isolation and identification of a novel fibrinolytic Bacillus tequilensis CWD-67 from dumping soils enriched with poultry wastes.

A newly isolated strain, CWD-67, which exhibited high fibrinolytic activity, was screened from dumping soils enriched with poultry wastes. The strain was identified as Bacillus tequilensis (KF897935) by 16Sr RNA gene sequence analysis and biochemical characterization. A fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate precipitation, membrane concentration, dialysis, ion-exchange, and gel filtration chromatography. SDS-PAGE analysis showed that the purified enzyme was a monomeric protein with an apparent molecular weight of 22 kDa, which is the lowest among Bacillus fibrinolytic enzymes reported to date. The purified enzyme was confirmed to have fibrinolytic activity by a fibrin zymogram. The optimal pH and temperature values of the enzyme were 8.0 and 45 °C, respectively. The enzyme was completely inhibited by PMSF and significantly inhibited by EDTA, TPCK, Co(2+), Zn(2+), and Cu(2+), suggesting a chymotrypsin-like serine metalloprotease. In vitro assays revealed that the purified enzyme could catalyze fibrin lysis effectively, indicating that this enzyme could be a useful fibrinolytic agent.