Environmental Sustainability and Physicochemical Property Screening of Chitin and Chitin-Glucan from 22 Fungal Species.

Thanks to its biobased character with embedded biogenic carbon, chitin can aid in the transition to a sustainable circular economy by replacing fossil carbon from the geosphere. However, meeting current demands for material availability and environmental sustainability requires alternative methods limiting conventional chemical and energy-consuming chitin extraction from crustaceans. To assist future chitinous bioproduct development, this work analyzes the physicochemical properties and potential environmental sustainability of fungal chitin-glucan complexes. A conventional isolation procedure using sodium hydroxide, a weak acid, and short reaction times are applied to the fruiting body of 22 fungal species. Besides, the valorization of underutilized waste streams including Agaricus bisporus and Agaricus brunnescens stipes is investigated. The carbohydrate analysis renders chitin fractions in the range of 9.5-63.5 wt %, while yields vary from 4.2 to 29.9%, and the N-acetylation degree in found in between 53.0 and 98.7%. The sustainability of the process is analyzed using life cycle assessment (LCA), providing impact quantification for global warming potential, terrestrial acidification, freshwater eutrophication, and water use. With 87.5-589.3 kg·CO2-equiv per kilo, potentially lower global warming potential values in comparison to crustacean chitin are achieved. The crystallinity degree ranged from 28 to 78%, while the apparent chitin crystalline size (L020) is between 2.3 and 5.4 nm. Ten of the species yield α-chitin coexisting with semicrystalline glucans. Zwitterionic properties are observed in aqueous solutions, shifting from cationic to anionic at pH 4.5. With its renewable carbon content, fungal chitin is an environmentally sustainable alternative for high-value applications due to its balance of minimal treatment, low carbon footprint, material renewability, ease of isolation, thermal stability, zwitterionic behavior, biodegradability, and noncytotoxicity.

Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi.

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents.

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.

Transcriptional basis of enhanced photoinduction of carotenoid biosynthesis at low temperature in the fungus Neurospora crassa.

Stimulation by light of carotenoid biosynthesis in the mycelia of the fungus Neurospora crassa starts with transient transcriptional induction of the structural genes of the pathway triggered by the White Collar photoreceptor complex. Most studies on this process were carried out under standard growth conditions, but photoinduced carotenoid accumulation is more efficient if the fungus is incubated at low temperatures, from 6 to 12 °C. We have investigated the transcriptional photoresponse at 8 °C of the genes for proteins that participate in the carotenoid pathway. Exposure to light pulses of different light intensities revealed higher sensitivity if the mycelia were subsequently incubated at 8 °C compared to 30 °C. Illumination of precooled mycelia resulted in delayed kinetics of mRNA accumulation for the structural genes, and high mRNA accumulation for a longer time. Additionally, after a light pulse, stronger reduction in mRNAs for carotenoid genes was observed at 30 °C compared to 8 °C. A similar pattern was found for mRNAs of the photoreceptor genes wc-1 and vvd, the latter involved in photoadaptation. These results suggest that the increased efficiency in carotenoid photoinduction at low temperature is due to the higher mRNA levels of the structural genes under these conditions.

Biochemical Characterization of the DASH-Type Cryptochrome CryD From Fusarium fujikuroi.

Proteins from the cryptochrome/photolyase family utilize UV-A, blue or even red light to achieve such diverse functions as repair of DNA lesions by photolyases and signaling by cryptochromes. DASH-type cryptochromes retained the ability to repair cyclobutane pyrimidine dimers (CPDs) in single-stranded DNA regions in vitro. However, most organisms possess conventional CPD photolyases responsible for repair of these lesions in vivo. Recent work showed that the DASH-type cryptochrome CryD plays a regulatory role in diverse light-dependent processes in Fusarium fujikuroi. Here, we report our in vitro studies on heterologously expressed FfCryD. The purified protein contains N(5) ,N(10) -methenyltetrahydrofolate and flavin adenine dinucleotide as cofactors. Photoreduction and DNA photorepair experiments confirmed that FfCryD is active in light-driven electron transfer processes. However, the protein showed comparable affinities for CPD-comprising and undamaged DNA probes. Surprisingly, after purification, full-length FfCryD as well as a truncated version containing only the PHR domain bound RNA which influenced their behavior in vitro. Moreover, binding of FfCryD to RNA indicates a putative role in RNA metabolism or in posttranscriptional control of gene expression.

The flavoproteins CryD and VvdA cooperate with the white collar protein WcoA in the control of photocarotenogenesis in Fusarium fujikuroi.

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor.

Light-mediated participation of the VIVID-like protein of Fusarium fujikuroi VvdA in pigmentation and development.

The phytopathogen Fusarium fujikuroi is a model fungus for the production of different secondary metabolites. These include the acidic xanthophyll neurosporaxanthin, produced through the activity of the enzymes encoded by the car genes, of which carRA and carB play a major role. Expression of these genes is induced by light but, in contrast to other fungi, the induction is not impaired under continuous illumination in null mutants of the F. fujikuroi wc-1-like gene, wcoA. Therefore, we investigate the role of other blue-light photoreceptors. Here we describe the identification, regulation and targeted mutation of the F. fujikuroi gene vvdA, homologous of the Neurospora crassa vivid (vvd) gene. As found for vvd in N. crassa, expression of vvdA in F. fujikuroi is strongly stimulated by light, an activation that is severely reduced in the wcoA mutants. Deletion of vvdA in F. fujikuroi results in a paler pigmentation under constant light, explained by a reduced carotenoid production, a regulatory effect opposite to the enhanced carotenoid accumulation characteristic of the vvd mutants of N. crassa. No major changes were appreciated in the transcriptional regulation of the car genes following exposure to light, but a noticeable reduction was found upon long-term incubation under constant illumination. Additionally, the vvdA mutants produce less conidia and their colonies exhibit morphological alterations under constant light, such as a more compact development of aerial mycelia and a more solid attachment to the agar surface. The results indicate that VvdA participates in the regulation by light of mycelial development and affects the accumulation of carotenoids but it is not responsible of the transcriptional photoadaptation of the car genes.

The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism.

Survival of micro-organisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. We previously described the gene cut-1 of Neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. Here we report on the functional analysis of cutA, the orthologous gene in the phytopathogenic fungus Fusarium fujikuroi. cutA mRNA levels increased transiently after exposure to 0.68 M NaCl and were reduced upon return to normal osmotic conditions; deletion of the gene resulted in a partial reduction in tolerance to osmotic stress. ΔcutA mutants contained much lower intracellular levels of glycerol than the wild-type, and did not exhibit the increase following hyper-osmotic shock expected from the high osmolarity glycerol (HOG) response. cutA is linked and divergently transcribed with the putative glycerol dehydrogenase gene gldB, which showed the same regulation by osmotic shock. The intergenic cutA/gldB regulatory region contains putative stress-response elements conserved in other fungi, and both genes shared other regulatory features, such as induction by heat shock and by illumination. Photoinduction was also observed in the HOG response gene hogA, and was lost in mutants of the white collar gene wcoA. Previous data on glycerol production in Aspergillus spp. and features of the predicted CutA protein lead us to propose that F. fujikuroi produces glycerol from dihydroxyacetone, and that CutA is the enzyme involved in the synthesis of this precursor by dephosphorylation of dihydroxyacetone-3P.

Light-dependent functions of the Fusarium fujikuroi CryD DASH cryptochrome in development and secondary metabolism.

DASH (Drosophila, Arabidopsis, Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome of Fusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of the cryD gene is induced by light in the wild type, but not in mutants of the "white collar" gene wcoA. Targeted ΔcryD mutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-type cryD allele. In addition to microconidia, the colonies of the ΔcryD mutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryD mutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryD mutants. The phenotypic alterations of the ΔcryD mutants were only noticeable in the light, as expected from the low expression of cryD in the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.