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In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

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BACKGROUND: Melanin is a natural pigment that can be applied in different fields such as medicine, environment, pharmaceutical, and nanotechnology. Studies carried out previously showed that the melanin produced by the mel1 mutant from Aspergillus nidulans exhibits antioxidant, anti-inflammatory, and antimicrobial activities, without any cytotoxic or mutagenic effect. These results taken together suggest the potential application of melanin from A. nidulans in the pharmaceutical industry. In this context, this study aimed to evaluate the effect of factors L-tyrosine, glucose, glutamic acid, L-DOPA, and copper on melanin production by the mel1 mutant and to establish the optimal concentration of these factors to maximize melanin production. RESULTS: The results showed that L-DOPA, glucose, and copper sulfate significantly affected melanin production, where L-DOPA was the only factor that exerted a positive effect on melanin yield. Besides, the tyrosinase activity was higher in the presence of L-DOPA, considered a substrate required for enzyme activation, this would explain the increased production of melanin in this condition. After establishing the optimal concentrations of the analyzed factors, the melanin synthesis was increased by 640% compared to the previous studies. CONCLUSIONS: This study contributed to elucidating the mechanisms involved in melanin synthesis in A. nidulans as well as to determining the optimal composition of the culture medium for greater melanin production that will make it possible to scale the process for a future biotechnological application.

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Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.

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The attachment of one or more ubiquitin molecules by SCF (Skp-Cullin-F-box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δfbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications.IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organism Aspergillus nidulans in the presence of glucose and the biotechnologically relevant plant polysaccharide xylan.

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The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans, in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications.

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Filamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.

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C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.

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Carbon catabolite repression (CCR) is a process that selects the energetically most favorable carbon source in an environment. CCR represses the use of less favorable carbon sources when a better source is available. Glucose is the preferential carbon source for most microorganisms because it is rapidly metabolized, generating quick energy for growth. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, a C2H2 finger domain DNA-binding protein. The aim of this work was to investigate the regulation of CreA and characterize its functionally distinct protein domains. CreA depends in part on de novo protein synthesis and is regulated in part by ubiquitination. CreC, the scaffold protein in the CreB-CreC deubiquitination (DUB) complex, is essential for CreA function and stability. Deletion of select protein domains in CreA resulted in persistent nuclear localization and target gene repression. A region in CreA conserved between Aspergillus spp. and Trichoderma reesei was identified as essential for growth on various carbon, nitrogen, and lipid sources. In addition, a role of CreA in amino acid transport and nitrogen assimilation was observed. Taken together, these results indicate previously unidentified functions of this important transcription factor. These novel functions serve as a basis for additional research in fungal carbon metabolism with the potential aim to improve fungal industrial applications.

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The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.

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RESUMOA pesquisa de produtos naturais benéficos à saúde humana vem crescendo nos últimos 20 anos. Considerando que as plantas de Aloe são amplamente utilizadas pela população humana, em geral de maneira terapêutica, o objetivo deste estudo foi avaliar os efeitos de Aloearborescens Miller e Aloe barbadensis Miller, sobre o desenvolvimento vegetativo de linhagens normais e mutantes de Aspergillus nidulans. Conídios da linhagem biA1methG1, MSE e CLB3 de A. nidulans, foram inoculados em meio completo sem (Controle) e com extratos das duas espécies incubados por 2, 4, 6 e 8 horas a 37ºC, no escuro. Foi analisado em microscópio óptico, 200 conídios de cada tratamento. Para o desenvolvimento das colônias, as linhagens foram inoculadas no centro das placas juntamente com o meio de cultura sólido e sobre a membrana de diálise, visando a medição do diâmetro e do peso. A análise estatística foi baseada no teste de Tukey e todos os procedimentos experimentais foram conduzidos em triplicata. Todas as linhagens apresentaram interferências positivas quando expostas às plantas de Aloe, porém, de maneira variada. Ambas as espécies aceleraram a germinação em todas as linhagens testadas e atuaram na redução significativa de conídios mortos e/ou malformados. Em relação ao desenvolvimento vegetativo, todos os dados referentes ao peso úmido e diâmetro corrigido dos tratamentos demonstraram progressos, contudo, a razão diâmetro/peso apresentou somente na linhagem MSE, ação favorável dos tratamentos naturais. As informações deste estudo sugerem benefícios de A. arborescens e A. barbadensis, justificando a importância e continuidade da investigação, para melhor elucidar os mecanismos de ação dessas plantas.

ABSTRACTThe researches about natural products that arebeneficial to human health have been growing over the past 20 years. Since Aloe plants are broadly used by the general population, frequently due to therapeutic reasons, the objective of this study was to evaluate the effects of Aloe arborescens Millerand Aloe barbadensis Miller on the vegetative growth of normal and mutant strains of Aspergillus nidulans. The conidia of thebiA1methG1, MSE and CLB3 strains of A. nidulans were inoculated in complete environment without (control) and with extracts of two species of Aloeincubated for 2, 4, 6 and 8 hours at 37˚C. 200 conidia were analyzed by optical microscopy. For the development of the colonies, the strains were inoculated in the center of the plates together with the solid environment of the cultivation and over the dialysis membrane for measuring the diameter and weighing. The statistical analysis was based on the Tukey test and all experimental procedures were performed in triplicate. All strains showed positive interference when exposed to Aloe plants, however, through different manners. Both species have accelerated the germination in all tested strains and acted in the significant reduction of dead and / or malformed conidia. Regarding the vegetative growth, all data related to wet weight and corrected diameter of the treatments revealed progress, however, the ratio diameter/weightpresented improvement only in the MSE lineage, favorable action of natural treatments. The information from this study suggest that A. arborescens and A. barbadensis are beneficial, thus justifying the importance of research maintenance in order to better elucidate the action mechanisms of these plants.

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