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.

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 CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 µg·mL-1 PatE protein in the culture medium with a purity just under 90%.

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.

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.