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.

Profiling of the ß-glucosidases identified in the genome of Penicillium funiculosum: insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with ß-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various ß-glucosidases present in the genome of the hypercellulolytic fungus Penicillium funiculosum using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight ß-glucosidase genes identified in the P. funiculosum genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome. Among the three secreted ß-glucosidases, two belonged to the GH3 family and one belonged to the GH1 family. Homology models of these proteins predicted a deep and narrow active site for the GH3 ß-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 ß-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum-secreted proteins showed high ß-glucosidase activities with prominent bands on the 4-methylumbelliferyl ß-D-glucopyranoside zymogram. To understand the contributory effects of each of the three secreted ß-glucosidases (PfBgls), the corresponding gene was deleted separately, and the effect of the deletion on the ß-glucosidase activity of the secretome was examined. Although not the most abundant, PfBgl3A was found to be one of the most important ß-glucosidases, as evidenced by a 42% reduction in ß-glucosidase activity in the ΔPfBgl3A strain. Our results advance the understanding of the genetic and biochemical nature of all ß-glucosidases produced by P. funiculosum and pave the way to design a superior biocatalyst for the hydrolysis of lignocellulosic biomass. IMPORTANCE Commercially available cellulases are primarily produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous ß-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including ß-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the ß-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight ß-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.

Genome editing in Penicillium funiculosum using in vitro assembled CRISPR-Cas9 ribonucleoprotein complexes.

The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in Penicillium funiculosum, which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with in vitro synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021).

Charge neutralization of lysine via carbamylation reveals hidden aggregation hot-spots in tau protein flanking regions.

Tau protein is found abundantly in neurofibrillary tangles in Alzheimer's disease (AD). The longest human tau isoform (2N4R) has 44 lysine residues. Several lysine-based post-translational modifications (PTMs) such as glycation, acetylation, ubiquitination, and sumoylation have been implicated not only in AD, but also in other tauopathies. Carbamylation is one such lysine neutralizing age-related nonenzymatic PTM which can modulate the aggregation propensity of tau. In this work, we have studied the aggregation potential of lysine-rich regions of tau upon carbamylation which do not aggregate in their native form. Using an array of biophysical and microscopic analyses, such as ThT kinetic assay, fluorescence microscopy, Congo red staining, and scanning electron microscopy, we demonstrate that peptides derived from four of five such regions exhibit robust fibrillar amyloid formation. These regions are found in the N-terminal projection domain that encompasses proline-rich domain (148-153 and 223-230), repeat domain R1 (253-260), as well as fibrillary core region (368-378), and can be described as hidden aggregation hot-spots which become activated upon carbamylation. We have further compared the impact of carbamylation with acetylation on the aggregation propensity of lysine-rich peptide (254 KKVAVV259 ) using biophysical experiments and molecular dynamics simulations and deduced that carbamylation is a much stronger driver of aggregation than acetylation. Our findings may offer more insight into amyloid fibrils' interaction with hidden aggregation-prone nucleating sequences that act as hot-spots for inducing tau fibrillation.

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228.

BACKGROUND: Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. RESULTS: Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. CONCLUSIONS: In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Effect of 11 months of yoga training on cardiorespiratory responses during the actual practice of Surya Namaskar.

BACKGROUND: Surya Namaskar (SN), a popular traditional Indian yogic practice, includes practicing 12 physical postures with alternate forward and backward bending movement of the body along with deep breathing maneuvers. The practice of SN has become popular among yoga practitioners and other fitness conscious people. The long-term effect of practicing SN and other yogic practices on cardiorespiratory responses during SN are lacking. AIM: The present study was conducted to study the effect of yogic training on various cardiorespiratory responses during the SN practice in yoga trainees after a time interval of 3, 6, and 11 months. MATERIALS AND METHODS: The present study was conducted on 9 healthy male Army soldiers who underwent training in various yoga postures including SN, meditation, and pranayama for 1 h daily for 11 months. First, second, and third phase of the study was conducted in the laboratory after completion of 3, 6, and 11 months of the yoga training. The participants performed SN along with other yogic practices in the laboratory as per their daily practice schedule. The cardiorespiratory responses of the volunteers were recorded during actual practice of SN. STATISTICAL ANALYSIS: One-way repeated measure ANOVA followed by Tukey HSD. RESULTS: Oxygen consumption and heart rate during actual practice of SN was 0.794 ± 0.252, 0.738 ± 0.229, and 0.560 ± 0.165 L/min and 92.1 ± 11.6, 97.9 ± 7.3 and 87.4 ± 9.2 beats/min respectively at 1(st) , 2(nd) , and 3(rd) phase of yoga training. Minute ventilation and tidal volume also reduced from 19.9 ± 4.65 to 17.8 ± 4.41 L/min and 1.091 ± 0.021 to 0.952 L/breath from 1(st) phase to 3(rd) phase of yoga training. However, respiratory parameters like breathing rate (fR) did not show any reduction across the three phases. CONCLUSION: The results of the present study indicated that yogic training caused conditioning of cardiorespiratory parameters except fR, which did not reduce across three phases of training.

Comparison of cardiorespiratory responses between Surya Namaskar and bicycle exercise at similar energy expenditure level.

Surya Namaskar (SN), a popular traditional Indian yogic practice called "Sun Salutations", includes practice of twelve physical postures involving alternate backward bending and forward bending postures. The practice of twelve postures in succession makes one round of its practice. Many people practise it as part of their daily physical fitness regimen. No study is available to compare cardiorespiratory responses of SN with bicycle exercise (BE). 20 healthy Yoga instructors practicing various Yogic practices including SN since last 7-8 years participated in the study. They performed SN in the laboratory according to their customary daily practice routine. The subject also performed incremental load bicycle exercise test till exhaustion on their second visit for measuring their VO2 max. SN and BE were compared at three similar exercise intensity levels in terms of % of VO2 max. The exercise intensities were light (10-20% VO2 max), moderate (21-40% VO2 max) and high intensities (41-50% VO2 max). Heart rate at high work intensity was significantly higher in BE than SN (P < .001). Ventilation and carbon dioxide output were significantly higher in BE than SN at high exercise intensity (P < 0.001). Overall, cardiorespiratory stress is less in SN than BE at similar work intensities.