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Endolichenic fungi (ELF) have been recognized as a promising source of bioactive secondary metabolites. Sri Lanka, a biodiversity hotspot, harbours a remarkable diversity in mangrove ecosystems. In the present study, endolichenic fungi obtained from mangrove lichens of Puttalam lagoon were studied for their secondary metabolites. An endolichenic fungus, Neurospora ugadawe, isolated from the lichen host Graphis tsunodae Zahlbr. was identified up to the species level. Secondary metabolites of N. ugadawe were extracted into ethyl acetate and crude extract showed promising antioxidant and anti-inflammatory activities. Bioassay guided separation on silica gel column chromatography and preparative TLC were performed to isolate the bioactive pure compounds. Two novel bioactive pure compounds were identified as Neurosporalol 1 and Neurosporalol 2. Neurosporalol 1 showed the highest antioxidant activity compared with the positive control BHT. Further, this compound showed very low anti-inflammatory activity compared to that of the positive control aspirin. Neurosporalol 2 showed comparable antioxidant and moderate anti-inflammatory activities.

Nitric-oxide synthase activity was detected in Neurospora crassa cells with the measurement of the conversion of 3H-L-arginine to 3H-L-citrulline. Some characteristics of this activity (sensitivity to calcium ions, action of specific enzyme inhibitors, and western blot analysis) were similar to those of an inducible enzyme found in mammals. According to western blotting, the molecular weight of NO synthase was around 130 kDa. No light-dependent changes in the specific activity of NO synthase were revealed in the photocarotenogenesis and photoconidiation of N. сrassa.

Many fungal diseases remain poorly addressed by public health authorities, despite posing a substantial threat to humans, animals, and plants. More worryingly, few classes of anti-fungals have been developed to combat fungal infections thus far. These medications also have certain drawbacks in terms of toxicity, spectrum of activity, and pharmacokinetic properties. Hence, there is a dire need for discovery of novel anti-fungal agents. Melittin, the main constituent in the venom of European honeybee Apis mellifera, has attracted considerable attention among researchers owing to its potential therapeutic applications. To our knowledge, there has been no review pertinent to anti-fungal properties of melittin, prompting us to synopsize the results of experimental investigations with a special emphasis upon underlying mechanisms. In this respect, melittin inhibits a broad spectrum of fungal genera including Aspergillus, Botrytis, Candida, Colletotrichum, Fusarium, Malassezia, Neurospora, Penicillium, Saccharomyces, Trichoderma, Trichophyton, and Trichosporon. Melittin hinders fungal growth by several mechanisms such as membrane permeabilization, apoptosis induction by reactive oxygen species-mediated mitochondria/caspase-dependent pathway, inhibition of (1,3)-β-d-glucan synthase, and alterations in fungal gene expression. Overall, melittin will definitely open up new avenues for various biomedical applications, from medicine to agriculture. • Venom-derived peptides have potential for development of anti-microbial agents. • Many fungal pathogens are susceptible to melittin at micromolar concentrations. • Melittin possesses multi-target mechanism of action against fungal cells.

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-A resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

International audience; Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.

Temperature compensation is a fundamental property of all circadian clocks; temperature compensation results in a relatively constant period length at different physiological temperatures, but its mechanism is unclear. Formation of a stable complex between clock proteins and casein kinase 1 (CK1) is a conserved feature in eukaryotic circadian mechanisms. Here, we show that the FRQ-CK1 interaction and CK1-mediated FRQ phosphorylation, not FRQ stability, are main mechanisms responsible for the circadian temperature compensation phenotypes in Neurospora. Inhibition of CK1 kinase activity impaired the temperature compensation profile. Importantly, both the loss of temperature compensation and temperature overcompensation phenotypes of the wild-type and different clock mutant strains can be explained by temperature-dependent alterations of the FRQ-CK1 interaction. Furthermore, mutations that were designed to specifically affect the FRQ-CK1 interaction resulted in impaired temperature compensation of the clock. Together, these results reveal the temperature-compensated FRQ-CK1 interaction, which results in temperature-compensated CK1-mediated FRQ and WC phosphorylation, as a main biochemical process that underlies the mechanism of circadian temperature compensation in Neurospora. IMPORTANCE Temperature compensation allows clocks to adapt to all seasons by having a relatively constant period length at different physiological temperatures, but the mechanism of temperature compensation is unclear. Stability of clock proteins was previously proposed to be a major factor that regulated temperature compensation. In this study, we showed that the interaction between CK1 and FRQ, but not FRQ stability, explains the circadian temperature compensation phenotypes in Neurospora. This study uncovered the key biochemical mechanism responsible for temperature compensation of the circadian clock and further established the mechanism for period length determination in Neurospora. Because the regulation of circadian clock proteins by CK1 and the formation of a stable clock complex with CK1 are highly conserved in eukaryotic clocks, a similar mechanism may also exist in animal clocks.

Recent attempts have been made to develop a thermophilic composting process for organic sludge to not only produce organic fertilizers and soil conditioners, but to also utilize the generated ammonia gas to produce high value-added algae. The hydrolysis of organic nitrogen in sludge is a bottleneck in ammonia conversion, and its improvement is a major challenge. The present study aimed to elucidate the effects of inoculated Neurospora sp. on organic matter decomposition and ammonia conversion during thermophilic composting of two organic sludge types: anaerobic digestion sludge and shrimp pond sludge. A laboratory-scale sludge composting experiment was conducted with a 6-day pretreatment period at 30 °C with Neurospora sp., followed by a 10-day thermophilic composting period at 50 °C by inoculating the bacterial community. The final organic matter decomposition was significantly higher in the sludge pretreated with Neurospora sp. than in the untreated sludge. Correspondingly, the amount of non-dissolved nitrogen was also markedly reduced by pretreatment, and the ammonia conversion rate was notably improved. Five enzymes exhibiting high activity only during the pretreatment period were identified, while no or low activity was observed during the subsequent thermophilic composting period, suggesting the involvement of these enzymes in the degradation of hardly degradable fractions, such as bacterial cells. The bacterial community analysis and its function prediction suggested the contribution of Bacillaceae in the degradation of easily degradable organic matter, but the entire bacterial community was highly incapable in degrading the hardly degradable fraction. To conclude, this study is the first to demonstrate that Neurospora sp. decomposes those organic nitrogen fractions that require a long time to be decomposed by the bacterial community during thermophilic composting.

Repeat-induced point mutation (RIP) is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. RIP detects duplications irrespective of their origin, particular sequence, coding capacity, or genomic positions. Previous studies suggested that RIP involves a cardinally new mechanism of sequence recognition that operates on intact double-stranded DNAs. In the fungus Neurospora crassa, RIP can be mediated by a putative C5-cytosine methyltransferase (CMT) RID or/and a canonical CMT DIM-2. These distinct RIP pathways feature opposite substrate preferences: RID-dependent RIP is largely limited to the duplicated sequences, whereas DIM-2-dependent RIP preferentially mutates adjacent non-repetitive regions. Using DIM-2-dependent RIP as a principal readout of repeat recognition, here we show that GC-rich repeats promote stronger RIP compared to AT-rich repeats (independently of their intrinsic propensities to become mutated), with the relative contribution of AT base-pairs being close to zero. We also show that direct repeats promote much more efficient DIM-2-dependent RIP than inverted repeats; both the spacer DNA between the repeat units (the linker) and the flanking regions are similarly affected by this process. These and other results support the idea that repeat recognition for RIP involves formation of many short interspersed quadruplexes between homologous double-stranded DNAs, which need to undergo concomitant changes in their linking number to accommodate pairing.SUMMARYDuring repeat-induced point mutation (RIP) gene-sized duplications of genomic DNA are detected by a mechanism that likely involves direct pairing of homologous double-stranded DNAs. We show that DIM-2-dependent RIP, triggered by closely-positioned duplications, is strongly affected by their relative orientations (direct versus inverted). We also show that GC-rich repeats promote RIP more effectively than AT-rich repeats. These results support a model in which homologous dsDNAs can pair by establishing interspersed quadruplex-based contacts with concomitant changes in their supercoiling status.

Fungi grow by apical extension of their hyphae. The continuous growth requires constant delivery of vesicles, which fuse with the membrane and secrete cell wall biosynthesis enzymes. The growth mechanism requires the fungal cytoskeleton and turgor pressure. In a recent study by Fukuda et al. (mBio 12:e03196-20, 2021, https://doi.org/10.1128/mBio.03196-20), hyphal growth was studied in microfluidic devices with channels smaller than the hyphal diameter. The authors discovered that fast-growing fungi like Neurospora crassa enter the channels, but hyphal tips become fragile and rupture frequently, whereas slower-growing fungi like Aspergillus nidulans adapt their hyphal diameter and grow without problems through the channels. This study suggests two different growth mechanisms and a tradeoff between hyphal plasticity and growth speed.

Purpose Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides. Method Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity. Result Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro. Conclusion This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

The role of nitric oxide in the photomorphogenesis of several Neurospora сrassa strains (the wild-type strain wt-987, the nit-2 mutant, which lacks nitrite and nitrate reductase, and the nit-6 mutant, which lacks nitrite reductase) was evaluated from the content of nitrate and nitrite, the final products of NO decomposition, in the mycelium and cultivation medium. Analysis of the dynamics of nitrite release from the mycelium of the N. crassanit-6 strain in the course of photostimulated conidiogenesis indicated the possible participation of the NO-generating mechanism in the fungal photosignal transduction. Light-regulated conidiation in N. crassa was inhibited by the introduction of S-nitrosoglutathione, a nitrogen oxide donor, to the cultivation medium, and stimulated by the introduction of L-nitroarginine, an inhibitor of NO synthase, which is inderect indicative of the role of NO in the process. However, the absence of $${\text{NO}}_{2}^{ - }$$ release during the photostimulated development of the protoperithecia (precursors of the female sexual structures) indicated a low probability of NO participation in sexual propagation of the fungus.

A new post-treatment method of fungal biomass after fermentation is revealed. The post-treatment strategy was utilized to produce pigments as an additional valuable metabolite. Post-treatment included incubation at 95% relative humidity where the effects of harvesting time, light, and temperature were studied. Pigment-producing edible filamentous fungus Neurospora intermedia cultivated on ethanol plant residuals produced 4 g/L ethanol and 5 g/L fungal biomass. Harvesting the pale biomass after 48 h submerged cultivation compared to 24 h or 72 h increased pigmentation in the post-treatment step with 35% and 48%, respectively. The highest pigment content produced, 1.4 mg/g dry fungal biomass, was obtained from washed biomass treated in light at 35 °C whereof the major impact on pigmentation was from washed biomass. Moreover, post-treated biomass contained 50% (w/w) crude protein. The post-treatment strategy successfully adds pigments to pre-obtained biomass. The pigmented fungal biomass can be considered for animal feed applications for domestic animals.

The nutrition quality and bioactivity of soybean meal was investigated via solid-state fermentation using Neurospora crassa (NC) alone or Neurospora crassa and saccharomyces cerevisiae (NCSC) together. The optimized fermentation conditions of NC or NCSC were obtained as follow: fermentation temperature (30 °C both), fermentation time (72 h vs 74.7 h), solid-liquid ratio [1:3.5 vs 1:3.0 (g/mL)], inoculation amount (the inoculation amount of fungi/soybean meal (dry weight)) of Neurospora crassa + Saccharomyces [1% (m/m)+0 vs 1% (m/m) +20% (v/m)], initial pH (7.0 both). The protein hydrolysis of fermented soybean meal (FSM) could reach to 10.05% and 8.05% by NC and NCSC, respectively. In fermented soybean meal by NC, the level of total free amino acids (TFAA) increased 13 times (p

Intracellular calcium signaling has been implicated in control of a variety of circadian processes in animals and plants but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock we screened knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. Loss of NCA-2 results in an increase in cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period as measured by both the core oscillator and overt clock. Genetic analyses showed that mutations in certain frq phospho-sites, and in Ca2+-calmodulin-dependent kinase (camk-2), are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca+2 leads to increased activity of CAMK-2 leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level some CAMKs undergo more auto-phosphorylations in Δnca-2, consistent with high calcium in the Δnca-2 mutant influencing the enzymatic activity of CAMKs. NCA-2 interacts with multiple proteins including CSP-6, a protein known to be required for circadian output. Most importantly, expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2, firmly places calcium signaling within the larger circadian system where it acts as both an input to and output from the core clock.ImportanceCircadian rhythms are based on cell-autonomous, auto-regulatory, feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. Building on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by loss of NCA-2 results in over-activation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, expression of NCA-2 is itself controlled by the molecular clock. In this way calcium signaling can be seen as providing both input to and output from the circadian system.

In vitro selection is an established approach to create artificial ribozymes with defined activities or to modify the properties of naturally occurring ribozymes. For the Varkud satellite ribozyme of Neurospora, an in vitro selection protocol based on its phosphodiester bond cleavage activity has not been previously reported. Here, we describe a simple protocol for cleavage-based in vitro selection that we recently used to identify variants of the Varkud satellite ribozyme able to target and cleave a non-natural stem-loop substrate derived from the HIV-1 TAR RNA. It allows quick selection of active ribozyme variants from the transcription reaction based on the size of the self-cleavage product without the need for RNA labeling. This results in a streamlined procedure that is easily adaptable to engineer ribozymes with new activities.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases. IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

International audience; The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing.

Two new α-pyrone derivatives, udagawanones A (1) and B (2), along with the known compounds (Z)-4-hydroxy-3-(3-hydroxy-3-methylbut-1-en-1-yl)benzoic acid (3), isosclerone (4), cyclo-(L-Leu-L-Pro) (5), and cyclo-(L-Pro-L-Tyr) (6), were isolated from cultures of the endophyte Neurospora udagawae. Their structures were elucidated by extensive spectroscopic methods and single crystal X-ray diffraction. Both compounds feature oxidized functionalities at the C-2 position not previously observed in other tetrasubstituted α-pyrones from fungi. Compound 1 exhibited moderate antibacterial (vs. Staphylococcus aureus) and antifungal (vs. Rhodoturula glutinis) activities and cytotoxicity against KB3.1 cells.

Neurospora crassa is a typical organism for many genetics study, DNA methylation is one of the most important process in DNA sequences which control the gene expression without changing DNA sequences. The Neurospora have a two genes dim-2 which is required for all known DNA methylation and rid-1 which is defeat any change in sequences. In silico program was used to analysis Neurospora DNA by designed two types of PCR primer, first set is for detection each gene and second set for detection CpG island in two types methylated and unmethylated primer. As well as using selective sensitive methylation restriction enzyme by draw an enzyme map by using Snapgene program tool.

International audience; DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivities of soil microbial processes. Enzymatic rates could increase at higher temperatures, but this response could change over time if soil microbes adapt to warming. We used the Arrhenius relationship, biochemical transition state theory, and thermal physiology theory to predict the responses of extracellular enzyme Vmax and Km to temperature. Based on these concepts, we hypothesized that Vmax and Km would correlate positively with each other and show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower Vmax , Km , and Km temperature sensitivity but higher Vmax temperature sensitivity. We tested these hypotheses with isolates of the filamentous fungus Neurospora discreta collected from around the globe and with decomposing leaf litter from a warming experiment in Alaskan boreal forest. For Neurospora extracellular enzymes, Vmax Q10 ranged from 1.48 to 2.25, and Km Q10 ranged from 0.71 to 2.80. In agreement with theory, Vmax and Km were positively correlated for some enzymes, and Vmax declined under experimental warming in Alaskan litter. However, the temperature sensitivities of Vmax and Km did not vary as expected with warming. We also found no relationship between temperature sensitivity of Vmax or Km and mean annual temperature of the isolation site for Neurospora strains. Declining Vmax in the Alaskan warming treatment implies a short-term negative feedback to climate change, but the Neurospora results suggest that climate-driven changes in plant inputs and soil properties are important controls on enzyme kinetics in the long term. Our empirical data on enzyme Vmax , Km , and temperature sensitivities should be useful for parameterizing existing biogeochemical models, but they reveal a need to develop new theory on thermal adaptation mechanisms.

Lignocellulosic biomass is an abundant renewable resource that can be used as a feedstock for production of second-generation biofuels. Currently, the bottleneck to generation of such fuels lies in the expensive and technically challenging process for converting biomass to fermentable sugars. Filamentous fungi are efficient at depolymerizing plant biomass. These fungi are also used for the production of industrial enzymes due to their ability to secrete large quantities of enzymes. The filamentous fungus Neurospora crassa is a genetically tractable model organism and a proficient degrader of plant biomass. In this dissertation, the enzymatic depolymerization of cellulose by Neurospora crassa was investigated using a combination of quantitative proteomics, genetics, and biochemistry. Chapter 1 is an introduction to lignocellulosic biomass and the organisms and enzymes that degrade it. In Chapter 2, a quantitative proteomic approach was taken to characterize the secretome of Neurospora crassa during growth on microcrystalline cellulose. Depolymerization of cellulose occurs by endoglucanases that hydrolyze internal glycosidic bonds and cellobiohydrolases that hydrolyze cellobiose from the reducing or non-reducing chain ends of cellulose. In addition to these canonical cellulases, a number of other proteins were quantified including a beta-glucosidase, a cellobiose dehydrogenase (CDH), and glycosyl hydrolase family 61 (GH61) enzymes. The beta-glucosidase and 3 cellulases were purified and biochemically characterized. While these 4 enzymes represent more than 85% of the cellulase in the secretome, they were significantly impaired in their rate of cellulose degradation relative to the complete set of enzymes in the secretome. This result suggested that proteins other than canonical cellulases may be important in cellulose depolymerization by fungi. The deletion of a gene encoding cellobiose dehydrogenase in N. crassa is described in Chapter 3. Cellobiose dehydrogenase (CDH) catalyzes the oxidation of cellobiose to cellobionolactone, but the biological function of this protein was previously unknown. Deletion of cdh-1 reduced cellulase activity 37-49% and addition of purified CDHs to the cdh-1 deletion strain resulted in a 1.6 to 2.0-fold stimulation in cellulase activity. The stimulatory effect of CDH required the presence of molecular oxygen and other secreted metalloproteins. The discovery that cellobiose dehydrogenase plays an integral role in degradation of cellulose marked a significant shift from previous models centered on the degradation of cellulose by mixtures of glycosyl hydrolases.To determine the molecular mechanisms by which CDH can enhance cellulase activity, a fractionation strategy was employed as described in Chapter 4 to identify synergistic metalloproteins in the N. crassa secretome. CDH was shown to enhance cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs), previously called GH61 enzymes. These enzymes catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage and facilitate elimination of the adjacent carbohydrate moiety. A further discussion of the mechanism of copper-dependent PMOs is discussed in Chapter 5. Here, the action of different PMOs was shown to be regiospecific resulting in oxidized products modified at C1 on the reducing end or C4 on the non-reducing end. CDHs and proteins related to the PMOs are found in cellulolytic species throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose depolymerization and could significantly reduce the cost of biofuel production.In chapter 6, the development of N. crassa as a host for recombinant expression of secreted enzymes is described. Expression of the endocellulase GH5-1 is used to complement the phenotype of a gh5-1 deletion strain. Experiments with GH5-1 fused to GFP (green fluorescent protein) were used to visualize the binding of this endocellulase to plant biomass. Additional applications for N. crassa as an expression host for fundamental studies of biomass depolymerizing enzymes are described.