Biology subjects

Yazdani, S. S.

Publications and source records attributed to Yazdani, S. S..

5 recordsLinked to original sources

bioRxiv2026

Synthetic lethality of fungal catabolite repressors reveals essential coupling of carbon repression with mitochondrial and ribosomal gene expression

Carbon catabolite repression (CCR) enables fungi to preferentially utilize favorable carbon sources through global transcriptional control. In filamentous fungi, CCR is classically attributed to the C2H2 zinc-finger repressor Mig1/CreA; however, residual repression following Mig1 disruption suggests additional regulatory layers in Talaromyces pinophilus (previously called Penicillium funiculosum NCIM1228). Here, we identify Mig3, a previously uncharacterized, Eurotiales-specific C2H2 transcription factor that functions redundantly with Mig1 in CCR. Mig3 contains conserved N-terminal zinc-finger motifs and a fungal transcription factor middle homology region and binds canonical Mig1 response elements with higher DNA affinity. Genetic analyses reveal that simultaneous loss of Mig1 and Mig3 is synthetically lethal, indicating an essential shared function beyond carbon repression. Integrative transcriptomic, chromatin immunoprecipitation sequencing, and proteomic analyses demonstrate that Mig1 and Mig3 directly regulate genes encoding mitochondrial and ribosomal components, including key factors involved in oxidative phosphorylation, while repressing genes required for alternative carbon metabolism. Loss of either repressor compromises expression of respiratory and ribosomal genes and confers hypersensitivity to mitochondrial and ribosomal inhibitors. In addition, both repressors physically associate with mitochondrial, ribosomal, and central metabolic proteins, suggesting coordination of transcriptional and post-transcriptional regulatory mechanisms. Together, our findings redefine fungal catabolite repressors as metabolic gatekeepers that couple CCR to mitochondrial and ribosome biogenesis, thereby controlling the metabolic switch between core cellular growth processes and enzyme secretion.

microbiology↗

bioRxiv2025

Changes in biophysical characteristics of YghA from E. coli due to variation in pH

YghA from E. coli has been reported to be involved in conferring tolerance against furan aldehyde inhibitors which are commonly generated in thermo-acidic pretreatment of lignocellulosic biomass. In this study we report the biophysical characterization of YghA from E. coli as a function of variation in pH. Fluorescence intensity of YghA increased by 2.5-fold in acidic pH as compared to circumneutral pH. In presence of the hydrophilic 8-anilinonaphthalene-1-sulfonic acid (ANS) dye, a 68 - 79.4-fold increase in fluorescence intensity at acidic pH was observed as compared to circumneutral pH, while at basic pH the increase was only 1 to 2-fold. Secondary structure analysis by circular dichroism signal at 222 and 208 nm suggests that the secondary structure of YghA is primarily composed of alpha-helix at pH 7 and the secondary structure is abruptly lost at pH 3 and lower. In agreement with these observations, MD simulations predicted greater structural variations at low pH when compared to neutral or high pH. Interface energy calculations using computational docking protocols suggested that YghA forms relatively more stable complex with NADH. The in silico pulling assay results also show that NADH is more preferred compared to NADPH. Molecular dynamics simulations also indicate that YghA is structurally unstable at acidic pH with significant variations in the root mean square deviation values of the tetramer backbone. Residues GLU 84 at pH 7 together with PRO 24 and LEU 236 at pH 1 were identified as flexible residues and are promising target for future mutagenesis studies targeted towards improving structural stability of YghA.

biochemistry↗

bioRxiv2023

Mechanistic insights into the role of Ca2+-stimulated AMPK in the secretion of cellulases during carbon stress

The response of filamentous fungi towards recalcitrant carbohydrates is majorly governed by transcriptional activators of cellulase genes; however, little is known about the downstream events beyond transcription. We show here in Penicillium funiculosum that increasing the expression of a transcriptional activator CLR-2 in the catabolically derepressed strain, {Delta}Mig1, didnt exhibit a synergistic effect on cellulase production unless Ca2+ was simultaneously increased. The RNA-seq screen for Ca2+-activated kinases identified SNF1-AMPK and SSP1-AMPKK as being specific to cellulose induction. Deletion of snf1 led to negligible secretion of cellulase upon induction. Quantitative whole-cell proteomics followed by chemical-genetic experiments with snf1-deleted strain showed that Ca2+-signaling channelizes carbon, nitrogen and energy sources towards cellulase production. Further, Ca2+-signaling phosphorylates SNF1-AMPK via SSP1, which in turn downregulates the phospho-HOG1 levels, leading to stimulus for cellulase secretion during carbon stress. The findings reported here are significant for understanding fungal pathology and developing second-generation biorefineries.

cell biology↗

bioRxiv2022

Profiling of the β-glucosidases identified in the genome of Penicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Enzymatic lignocellulosic biomass conversion to bioethanol is dependent on efficient enzyme systems with {beta}-glucosidase as a key component. In this study, we performed in-depth profiling of the various {beta}-glucosidases present in the genome of the hypercellulolytic fungus; Penicillium funiculosum using genomics, transcriptomics, proteomics and molecular dynamics simulation approaches. Of the eight {beta}-glucosidase genes identified in the P. funiculosum genome, three were found to be extracellular, as evidenced by presence of signal peptides and mass spectrometry. Among the three secreted {beta}-glucosidase, two belonged to the GH3 and one belonged to GH1 families. Modelled structures of these proteins predicted a deep and narrow active site for the GH3 {beta}-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 {beta}-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum secretome showed high {beta}-glucosidase activities with prominent bands on 4-methylumbelliferyl {beta}-D-glucopyranoside (MUG) zymogram. To understand the contributory effect of each of the three secreted {beta}-glucosidases (PfBgls), the corresponding gene was deleted separately and the effect of the deletion on {beta}-glucosidase activity of the secretome was examined. Although not the most abundant {beta}-glucosidase, PfBgl3A was found to be the most significant one as evidenced by a 42 % reduction in {beta}-glucosidase activity in the {Delta}PfBgl3A strain. To improve the thermostability, two mutants of PfBgl3A were designed with the help of molecular dynamics (MD) simulation and were expressed in Pichia pastoris for evaluation. The PfBgl3A mutant (Mutant A) gave 1.4 fold increase in the half-life (T1/2) of the enzyme at 50{degrees}C. IMPORTANCECommercially available cellulases are majorly produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous {beta}-glucosidase is often required to achieve desired optimal saccharification of cellulosic feedstocks. This challenge has led to exploration of other cellulase-producing strains because of the importance of this class of enzymes in the cellulose deconstruction machinery. The non-model hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases. Various genetic interventions targeted at strain improvement for cellulase production have been performed. However, the {beta}-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all the eight {beta}-glucosidases of P. funiculosum via molecular and computational approaches and enhancing thermostability of the most promising {beta}-glucosidase via protein engineering. The results of this study set the background for future engineering strategies to transform the fungus into an industrial workhorse.

microbiology↗

bioRxiv2020

Insights into structure of Penicillium funiculosum LPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess auxiliary activity are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding affinity with a {Delta}G free energy of -46 k kcal/mol when compared with that of TaLPMO9A (-31 kcal/mol). The enzyme was further found to be highly thermostable at elevated temperature with a half-life of [~]88 h at 50 {degrees}C. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress this LPMO and Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, PfMig188, in order to improve its saccharification performance towards acid pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed [~]200% and [~]66% increase in LPMO and Avicelase activities, respectively. While the secretomes of individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 6% and 13%, respectively, over PfMig188 at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency over PfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading. ImportanceEnzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation bio-based industries. While efforts are being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge confronting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to get an enzymatic cocktail with balanced activity from a single host. This report provides for the first time the annotation and structural analysis of an uncharacterized thermostable lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in biomass deconstruction upon overexpression in catabolite derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed simultaneously with the LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities with the corresponding improvement in its saccharification performance at high substrate loading by [~]20% using a minimal amount of protein.

microbiology↗