Expanded Gene Regulatory Network Reveals Potential Light-Responsive Transcription Factors and Target Genes in Cordyceps militaris.

Cordyceps militaris, a fungus widely used in traditional Chinese medicine and pharmacology, is recognized for its abundant bioactive compounds, including cordycepin and carotenoids. The growth, development, and metabolite production in various fungi are influenced by the complex interactions between regulatory cascades and light-signaling pathways. However, the mechanisms of gene regulation in response to light exposure in C. militaris remain largely unexplored. This study aimed to identify light-responsive genes and potential transcription factors (TFs) in C. militaris through an integrative transcriptome analysis. To achieve this, we reconstructed an expanded gene regulatory network (eGRN) comprising 507 TFs and 8662 regulated genes using both interolog-based and homolog-based methods to build the protein-protein interaction network. Aspergillus nidulans and Neurospora crassa were chosen as templates due to their relevance as fungal models and the extensive study of their light-responsive mechanisms. By utilizing the eGRN as a framework for comparing transcriptomic responses between light-exposure and dark conditions, we identified five key TFs-homeobox TF (CCM_07504), FlbC (CCM_04849), FlbB (CCM_01128), C6 zinc finger TF (CCM_05172), and mcrA (CCM_06477)-along with ten regulated genes within the light-responsive subnetwork. These TFs and regulated genes are likely crucial for the growth, development, and secondary metabolite production in C. militaris. Moreover, molecular docking analysis revealed that two novel TFs, CCM_05727 and CCM_06992, exhibit strong binding affinities and favorable docking scores with the primary light-responsive protein CmWC-1, suggesting their potential roles in light signaling pathways. This information provides an important functional interactive network for future studies on global transcriptional regulation in C. militaris and related fungi.

Convergent evolution links molybdenum insertase domains with organism-specific sequences.

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Adaptative responses of Neurospora crassa by histidine kinases upon the attack of the arthropod Sinella curviseta.

Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of ß-glucan, ergothioneine, ergosterol, and carotenoids. ß-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.

As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.

VE-1 regulation of MAPK signaling controls sexual development in Neurospora crassa.

Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa, this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2, but not lae-1, leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2, suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/VE-2 complex is a key element in transcriptional regulation that occurs during sexual development. IMPORTANCE: Sexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa. VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes.

A novel luciferase-based reporter tool to monitor the dynamics of carbon catabolite repression in filamentous fungi.

Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours.

H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora.

Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11 phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation) and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.

Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation.

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.

Utilizing Deep Neural Networks to Fill Gaps in Small Genomes.

With the widespread adoption of next-generation sequencing technologies, the speed and convenience of genome sequencing have significantly improved, and many biological genomes have been sequenced. However, during the assembly of small genomes, we still face a series of challenges, including repetitive fragments, inverted repeats, low sequencing coverage, and the limitations of sequencing technologies. These challenges lead to unknown gaps in small genomes, hindering complete genome assembly. Although there are many existing assembly software options, they do not fully utilize the potential of artificial intelligence technologies, resulting in limited improvement in gap filling. Here, we propose a novel method, DLGapCloser, based on deep learning, aimed at assisting traditional tools in further filling gaps in small genomes. Firstly, we created four datasets based on the original genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla. To further extract effective information from the gene sequences, we also added homologous genomes to enrich the datasets. Secondly, we proposed the DGCNet model, which effectively extracts features and learns context from sequences flanking gaps. Addressing issues with early pruning and high memory usage in the Beam Search algorithm, we developed a new prediction algorithm, Wave-Beam Search. This algorithm alternates between expansion and contraction phases, enhancing efficiency and accuracy. Experimental results showed that the Wave-Beam Search algorithm improved the gap-filling performance of assembly tools by 7.35%, 28.57%, 42.85%, and 8.33% on the original results. Finally, we established new gap-filling standards and created and implemented a novel evaluation method. Validation on the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla showed that DLGapCloser increased the number of filled gaps by 8.05%, 15.3%, 1.4%, and 7% compared to traditional assembly tools.

Remodeling of perturbed chromatin can initiate de novo transcriptional and post-transcriptional silencing.

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.

Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10.

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

The RPD3L deacetylation complex is required for facultative heterochromatin repression in Neurospora crassa.

Repression of facultative heterochromatin is essential for developmental processes in numerous organisms. Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes. Although this methylation is frequently associated with silencing, the detailed mechanism of repression remains incompletely understood. We utilized a forward genetics approach to identify genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa and identified three previously uncharacterized genes that are important for silencing: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007), and rlp2 (RPD3L protein 2; NCU02898). We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae Rpd3L complex and are required for repression of a subset of H3K27-methylated genes. Deletion of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at up-regulated genes, suggesting that RPD3L-driven deacetylation is a factor required for silencing of facultative heterochromatin in N. crassa, and perhaps in other organisms.

The clock in growing hyphae and their synchronization in Neurospora crassa.

Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device.

Acetylation of WCC is dispensable for the core circadian clock but differentially regulates acute light responses in Neurospora.

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is mainly controlled by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

Functions and mechanisms of A-to-I RNA editing in filamentous ascomycetes.

Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock.

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

Natural oscillations known as circadian rhythms influence many processes in humans and other animals including sleep, eating, brain activity and body temperature. These rhythms allow us to anticipate and prepare for regular changes in our environment including day-night cycles and the temperature of our surroundings. Circadian clocks in animals, fungi and other 'eukaryotic' organisms rely on networks of components that repress their own production to generate oscillations in their levels in cells over the course of a 24-hour period. The components in animal and fungus circadian clocks are different but there are strong similarities in their properties and how the networks operate. As a result, a type of fungus known as Neurospora crassa is often used as a model to study how circadian rhythms work in animals. A central component in the N. crassa circadian clock is a protein known as Frequency (FRQ). It is a large protein that, unlike most proteins, lacks a well-defined, three-dimensional structure. Despite this, it is able to bind to and regulate other proteins to repress its own production. One of its protein partners known as CK1 attaches small tags known as phosphate groups to FRQ to set the length of the circadian rhythm. However, it remains unclear how FRQ interacts with its protein partners or what effect the phosphate groups have on its activity. To address this question, Tariq, Maurici et al. used biochemical approaches to study the structure of FRQ. The experiments revealed that it contains a compact core that is able to bind to CK1 and other protein partners. The way FRQ regulates its protein partners is unusual: it undergoes a chemical process known as liquid-liquid phase separation to sequester other circadian clock proteins and modulate their enzymatic activities. In this process, a solution containing molecules of FRQ separates into two distinct components (known as phases), one of which contains FRQ and its partners in a concentrated liquid-like mixture. Evidence for such mixtures has also been found in living fungal cells. Further experiments suggest that liquid-liquid phase separation of FRQ may allow the clock to compensate for changes in temperature to maintain a regular rhythm. The circadian clocks of animals and other organisms all have proteins that perform similar roles as FRQ and maintain sequence properties that promote liquid-liquid phase separation. Therefore, it is possible that liquid-liquid phase separation may be a common feature of circadian rhythms in nature.

CDF family of zinc transporters ZRC-1, MSC-2, and ZRG-17 are involved in survival at high zinc conditions, vegetative development, and cellulase utilization in Neurospora crassa.

The cation diffusion facilitator (CDF) family of zinc transporters plays a crucial role in zinc homeostasis in eukaryotes, including fungi. Here, we investigated the cell functions and genetic interactions of CDF zinc transporters zrc-1 and msc-2 in Neurospora crassa. The Δzrc-1 mutant could not grow in a high-zinc environment, indicating that the zinc transporter protein ZRC-1 was essential for growth in high-zinc conditions. However, the deletion of msc-2 did not show any severe phenotypic defects. Furthermore, we studied the genetic interactions of the zinc transporters using the CDF double mutants. Previously, zrg-17 was reported to be critical, where the Δzrg-17 mutant showed defects in both vegetative development and asexual sporulation. Interestingly, the Δmsc-2;Δzrg-17 double mutant showed phenotypes similar to the wild type, and restored the phenotypic defects of the Δzrg-17 mutation. However, the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants continue to display phenotypic defects like their parental single mutants. The double mutant Δzrc-1;Δzrg-17 showed severe vegetative growth defects, including slow growth, short aerial hyphae, narrowed septation, and defective asexual sporulation. In addition, aerial hyphae development of the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants were reduced under endoplasmic reticulum stress. Thus, this study revealed the cell functions and genetic interactions of zrc-1, msc-2, and zrg-17 for vegetative development and tolerance to stress conditions in N. crassa.

Conversion of Deproteinized Cheese Whey to Lactobionate by an Engineered Neurospora crassa Strain F5.

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven ß-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3 µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37 g/L of LBA from 45 g/L of lactose in 27 h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37 g/L/h.

A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.