Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

Transcriptome analysis of Aspergillus niger xlnR and xkiA mutants grown on corn Stover and soybean hulls reveals a highly complex regulatory network.

BACKGROUND: Enzymatic plant biomass degradation by fungi is a highly complex process and one of the leading challenges in developing a biobased economy. Some industrial fungi (e.g. Aspergillus niger) have a long history of use with respect to plant biomass degradation and for that reason have become 'model' species for this topic. A. niger is a major industrial enzyme producer that has a broad ability to degrade plant based polysaccharides. A. niger wild-type, the (hemi-)cellulolytic regulator (xlnR) and xylulokinase (xkiA1) mutant strains were grown on a monocot (corn stover, CS) and dicot (soybean hulls, SBH) substrate. The xkiA1 mutant is unable to utilize the pentoses D-xylose and L-arabinose and the polysaccharide xylan, and was previously shown to accumulate inducers for the (hemi-)cellulolytic transcriptional activator XlnR and the arabinanolytic transcriptional activator AraR in the presence of pentoses, resulting in overexpression of their target genes. The xlnR mutant has reduced growth on xylan and down-regulation of its target genes. The mutants therefore have a similar phenotype on xylan, but an opposite transcriptional effect. D-xylose and L-arabinose are the most abundant monosaccharides after D-glucose in nearly all plant-derived biomass materials. In this study we evaluated the effect of the xlnR and xkiA1 mutation during growth on two pentose-rich substrates by transcriptome analysis. RESULTS: Particular attention was given to CAZymes, metabolic pathways and transcription factors related to the plant biomass degradation. Genes coding for the main enzymes involved in plant biomass degradation were down-regulated at the beginning of the growth on CS and SBH. However, at a later time point, significant differences were found in the expression profiles of both mutants on CS compared to SBH. CONCLUSION: This study demonstrates the high complexity of the plant biomass degradation process by fungi, by showing that mutant strains with fairly straightforward phenotypes on pure mono- and polysaccharides, have much less clear-cut phenotypes and transcriptomes on crude plant biomass.

The White-Rot Basidiomycete Dichomitus squalens Shows Highly Specific Transcriptional Response to Lignocellulose-Related Aromatic Compounds.

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.

Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

BACKGROUND: The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. RESULTS: The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. CONCLUSIONS: Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

High resolution visualization and exo-proteomics reveal the physiological role of XlnR and AraR in plant biomass colonization and degradation by Aspergillus niger.

In A. niger, two transcription factors, AraR and XlnR, regulate the production of enzymes involved in degradation of arabinoxylan and catabolism of the released l-arabinose and d-xylose. Deletion of both araR and xlnR in leads to reduced production of (hemi)cellulolytic enzymes and reduced growth on arabinan, arabinogalactan and xylan. In this study, we investigated the colonization and degradation of wheat bran by the A. niger reference strain CBS 137562 and araR/xlnR regulatory mutants using high-resolution microscopy and exo-proteomics. We discovered that wheat bran flakes have a 'rough' and 'smooth' surface with substantially different affinity towards fungal hyphae. While colonization of the rough side was possible for all strains, the xlnR mutants struggled to survive on the smooth side of the wheat bran particles after 20 and 40 h post inoculation. Impaired colonization ability of the smooth surface of wheat bran was linked to reduced potential of ΔxlnR to secrete arabinoxylan and cellulose-degrading enzymes and indicates that XlnR is the major regulator that drives colonization of wheat bran in A. niger.

Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin.

Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.

The transcriptional activator GaaR of Aspergillus niger is required for release and utilization of d-galacturonic acid from pectin.

We identified the d-galacturonic acid (GA)-responsive transcriptional activator GaaR of the saprotrophic fungus, Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome-wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell, and to induce the GA catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to the expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.

Genetic Interaction of Aspergillus nidulans galR, xlnR and araR in Regulating D-Galactose and L-Arabinose Release and Catabolism Gene Expression.

In Aspergillus nidulans, the xylanolytic regulator XlnR and the arabinanolytic regulator AraR co-regulate pentose catabolism. In nature, the pentose sugars D-xylose and L-arabinose are both main building blocks of the polysaccharide arabinoxylan. In pectin and arabinogalactan, these two monosaccharides are found in combination with D-galactose. GalR, the regulator that responds to the presence of D-galactose, regulates the D-galactose catabolic pathway. In this study we investigated the possible interaction between XlnR, AraR and GalR in pentose and/or D-galactose catabolism in A. nidulans. Growth phenotypes and metabolic gene expression profiles were studied in single, double and triple disruptant A. nidulans strains of the genes encoding these paralogous transcription factors. Our results demonstrate that AraR and XlnR not only control pentose catabolic pathway genes, but also genes of the oxido-reductive D-galactose catabolic pathway. This suggests an interaction between three transcriptional regulators in D-galactose catabolism. Conversely, GalR is not involved in regulation of pentose catabolism, but controls only genes of the oxido-reductive D-galactose catabolic pathway.

Regulation of plant biomass utilization in Aspergillus.

The ability of fungi to survive in every known biotope, both natural and man-made, relies in part on their ability to use a wide range of carbon sources. Fungi degrade polymeric carbon sources present in the environment (polysaccharides, proteins, and lignins) to use the monomeric components as nutrients. However, the available carbon sources vary strongly in nature, both between biotopes and in time. The degradation of polymeric carbon sources is mediated through the production of a broad range of enzymes, the production of which is tightly controlled by a network of regulators and linked to the activation of catabolic pathways to convert the released monomers. This review summarizes the knowledge of Aspergillus regulators involved in plant biomass utilization.

Protein kinase C beta in postischemic brain mitochondria.

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCß with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCß in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCß inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCß might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCßΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCß acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.

Neuroprotective potential of biphalin, multireceptor opioid peptide, against excitotoxic injury in hippocampal organotypic culture.

UNLABELLED: Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. FINDINGS: (1) 0.025-0.1 µM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria.

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Association of protein kinase C delta and phospholipid scramblase 3 in hippocampal mitochondria correlates with neuronal vulnerability to brain ischemia.

Recent findings support the idea that mitochondrial integrity plays an important role in the propagation of excitotoxic ischemic signal and PKC is implicated in the regulation of mitochondrial membranes properties. One of the targets of PKC delta is phospholipid scramblase 3 (PLSCR3), an enzyme responsible for cardiolipin translocation from the inner to outer mitochondrial membrane. To get an insight into in vivo mechanism by which PKC delta mediates ischemia/reperfusion injury of hippocampal neurons, we examined the effects of transient brain ischemia in gerbil on association of PKC delta with mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant regions, and interactions between PKC delta and PLSCR3. Postischemic, biphasic and brain region-specific translocation of PKC delta to mitochondria was observed. First peak was at 30-60 min of reperfusion and the second was observed after 72-96 h following ischemia. PKC delta was translocated to mitochondria only in CA1 region. The PLSCR3 mRNA and protein was detected in brain by RT-PCR and sequence analysis, Western blotting and immunocytochemistry in electron microscopy (EM). Co-immunoprecipitation and double-labeled immuno-EM showed association of PKC delta and PLSCR3 in postischemic CA1 mitochondria. Additionally, the amount of tBid associated with mitochondria was elevated 96 h following ischemia. Our data suggest that in the postischemic brain PKC delta co-localizes with PLSCR3 in mitochondria and this event might influence the mitochondrial membranes architecture and delayed neurons degeneration.

Kalirin-7, a protein enriched in postsynaptic density, is involved in ischemic signal transduction.

Regulators of mitogen activated protein kinases (MAPK) and c-Jun N-terminal/stress-activated kinase (JNK) include Rho-like small GTP-binding proteins and their regulators. SynGAP and kalirin-7 are postsynaptic density-enriched proteins identified through their interaction with Rho GTPases and PSD-95 scaffold protein. We examined immunoreactivity of SynGAP, kalirin-7, and PSD-95, phosphorylation of MAPK and JNK in control and postischemic hippocampus in gerbil model of transient forebrain ischemia. In normal brain higher amount of kalirin-7 but a lower amount of P-JNK was found in ischemia-resistant hippocampal area: CA2-3, DG than in ischemia-vulnerable CA1. After 5 min ischemia and 1 h reperfusion a decrease of P-ERK and increase of P-JNK were uniformly observed in the hippocampal parts. By contrast, the amount of kalirin-7 in CA2-3, DG reached 56% (P < 0.001) of control while was doubled in CA1. Oppositely, the immunoreactivity of SynGAP was increased in CA2-3, DG and reduced in CA1. Our data indicate that SynGAP and kalirin-7 take part in the regulation of ischemic signal transduction but the mechanism does not seem directly connected with the activation of MAPK and JNK.

Cytochrome c binds to inositol (1,4,5) trisphosphate and ryanodine receptors in vivo after transient brain ischemia in gerbils.

Previously we have shown that the biphasic efflux of mitochondrial protein cytochrome c to cytoplasm is one of the important events of the delayed postichemic neuronal death. We concluded that early and transient appearance of cytochrome c in cytoplasm of cells recovering after ischemia was decisive for initiation of the pathological signaling cascade leading to neuronal death, but the precise mechanism remained unknown. In vitro cytochrome c was identified as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis. Here we show that in vivo cytochrome c interacts with inositol (1,4,5) trisphosphate receptor type 1 in gerbil hippocampus subjected to transient brain ischemia and short reperfusion. Moreover, cytochrome c binds also to ryanodine receptor type 2, the role of which in postischemic neuronal death is suggested. The complexes could be coimmunoprecipitated by antibodies against any of the two proteins. Our data verified that the mechanism observed in vitro applies to the pathological in vivo situation.