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Ítem Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions(Wiley, 2021) Batista-García, Ramón; Peidro-Guzmán, Heidy; González-Abradelo, Deborah; Fernández-López, Maikel; Dávila-Ramos, Sonia; Aranda-Ballesteros, Elizabet; Ortega-García, Angélica; Lira, Verónica; Gunde-Cimerman, Nina; Ramírez-Pliego, Óscar; Schnabel, Denhi; Jiménez-Gómez, Irina; Sánchez-Carbente, María; Folch, Jorge Luis; Sánchez-Reyes, Ayixon; Kumar, Vaidyanathan Vinoth; Cabana, Hubert; Santana, María Angélica; Mouriño-Pérez, Rosa Reyna; Aréchiga, ElvaPolycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.Ítem Haloadaptative Responses of Aspergillus sydowii to Extreme Water Deprivation: Morphology, Compatible Solutes, and Oxidative Stress at NaCl Saturation(MDPI, 2020) Batista-García, Ramón; Jiménez-Gómez, Irina; Valdés-Muñoz, Gisell; Moreno-Perlin, Tonatiuh; Mouriño-Pérez, Rosa Reyna; Sánchez-Carbente, María; Folch, Jorge Luis; Pérez-Llano, Yordanis; Gunde-Cimerman, Nina; Sánchez-Castellanos, NildaWater activity (aw) is critical for microbial growth, as it is severely restricted at aw < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.Ítem Stress Reshapes the Physiological Response of Halophile Fungi to Salinity(MDPI, 2020) Batista-García, Ramón; Pérez-Llano, Yordanis; Rodríguez-Pupo, Eya Caridad; Druzhinina, Irina; Chenthamara, Komal; Cai, Feng; Gunde-Cimerman, Nina; Gostinčar, Cene; Kostanjšek, Rok; Folch, Jorge Luis; Sánchez-Carbente, MaríaBackground: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.Ítem Physcomitrium patens Infection by Colletotrichum gloeosporioides: Understanding the Fungal–Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing(MDPI, 2021) Batista-García, Ramón; Otero, Adriana; Pérez-Llano, Yordanis; Reboledo, Guillermo; Lira, Verónica; Padilla-Chacón, Daniel; Folch, Jorge Luis; Sánchez-Carbente, María; Ponce-de-León, InésAnthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.Ítem Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration(2022) Batista-García, Ramón; Jiménez-Gómez, Irina; Valdés-Muñoz, Gisell; Moreno-Ulloa, Aldo; Pérez-Llano, Yordanis; Moreno-Perlin, Tonatiuh; Silva-Jiménez, Hortencia; Barreto, Fernando; Sánchez-Carbente, María; Folch, Jorge Luis; Gunde-Cimerman, Nina; Lago, AsunciónAlthough various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multiomics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13M NaCl) to optimal salinity (1M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of β-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.Ítem Tracking gene expression, metabolic profiles, and biochemical analysis in the halotolerant basidiomycetous yeast Rhodotorula mucilaginosa EXF-1630 during benzo[a]pyrene and phenanthrene biodegradation under hypersaline conditions(Elsevier, 2021) Batista-García, Ramón; Martínez-Ávila, Liliana; Peidro-Guzmán, Heidy; Pérez-Llano, Yordanis; Moreno-Perlin, Tonatiuh; Sánchez-Reyes, Ayixon; Fernández-Silva, Arline; Folch, Jorge Luis; Cabana, Hubert; Gunde-Cimerman, Nina; Ángeles, Gabriela; Aranda-Ballesteros, ElizabetPolyaromatic phenanthrene (Phe) and benzo[a]pyrene (BaP) are highly toxic, mutagenic, and carcinogenic contaminants widely dispersed in nature, including saline environments. Polyextremotolerant Rhodotorula mucilaginosa EXF-1630, isolated from Arctic sea ice, was grown on a huge concentration range -10 to 500 ppm- of Phe and BaP as sole carbon sources at hypersaline conditions (1 M NaCl). Selected polycyclic aromatic hydrocarbons (PAHs) supported growth as well as glucose, even at high PAH concentrations. Initially, up to 40% of Phe and BaP were adsorbed, followed by biodegradation, resulting in 80% removal in 10 days. While extracellular laccase, peroxidase, and un-specific peroxygenase activities were not detected, NADPH-cytochrome c reductase activity peaked at 4 days. The successful removal of PAHs and the absence of toxic metabolites were confirmed by toxicological tests on moss Physcomitrium patens, bacterium Aliivibrio fischeri, human erythrocytes, and pulmonary epithelial cells (A549). Metabolic profiles were determined at the midpoint of the biodegradation exponential phase, with added Phe and BaP (100 ppm) and 1 M NaCl. Different hydroxylated products were found in the culture medium, while the conjugative metabolite 1-phenanthryl-b-D glucopyranose was detected in the medium and in the cells. Transcriptome analysis resulted in 870 upregulated and 2,288 downregulated transcripts on PAHs, in comparison to glucose. Genomic mining of 61 available yeast genomes showed a widespread distribution of 31 xenobiotic degradation pathways in different yeast lineages. Two distributions with similar metabolic capacities included black yeasts and mainly members of the Sporidiobolaceae family (including EXF-1630), respectively. This is the first work describing a metabolic profile and transcriptomic analysis of PAH degradation by yeast.Ítem Simple screening protocol for identification of potential mycoremediation tools for the elimination of polycyclic aromatic hydrocarbons and phenols from hyperalkalophile industrial effluents(Elsevier, 2017) Batista-García, Ramón; Kumar, Vaidyanathan Vinoth; Ariste, Arielle; Tovar-Herrera, Omar; Savary, Olivier; Peidro-Guzmán, Heidy; González-Abradelo, Deborah; Jackson, Stephen A.; Dobson, Alan D.W.; Sánchez-Carbente, María; Cabana, Hubert; Folch, Jorge Luis; Leduc, RolandA number of fungal strains belonging to the ascomycota, basidiomycota and zygomycota genera were subjected to an in vitro screening regime to assess their ligninolytic activity potential, with a view to their potential use in mycoremediation based strategies to remove phenolic compounds and polycyclic aromatic hydrocarbons (PAHs) from industrial wastewaters. All six basidiomycetes completely decolorized remazol brilliant blue R (RBBR), while also testing positive in both the guaiacol and gallic acid tests indicating good levels of lignolytic activity. All the fungi were capable of tolerating phenanthrene, benzo-α- pyrene, phenol and p-chlorophenol in agar medium at levels of 10 ppm. Six of the fungal strains, Pseudogymnoascus sp., Aspergillus caesiellus, Trametes hirsuta IBB 450, Phanerochate chrysosporium ATCC 787, Pleurotus ostreatus MTCC 1804 and Cadophora sp. produced both laccase and Mn peroxidase activity in the ranges of 200–560 U/L and 6–152 U/L, respectively, in liquid media under nitrogen limiting conditions. The levels of adsorption of the phenolic and PAHs were negligible with 99% biodegradation being observed in the case of benzo-α-pyrene, phenol and p chlorophenol. The aforementioned six fungal strains were also found to be able to effectively treat highly alkaline industrial wastewater (pH 12.4). When this wastewater was supplemented with 0.1 mM glucose, all of the tested fungi, apart from A. caesiellus, displayed the capacity to remove both the phenolic and PAH compounds. Based on their biodegradative capacity we found T. hirsuta IBB 450 and Pseudogymnoascus sp., to have the greatest potential for further use in mycoremediation based strategies to treat wastestreams containing phenolics and PAHs.Ítem First demonstration that ascomycetous halophilic fungi (Aspergillus sydowii T and Aspergillus destruens) are useful in xenobiotic mycoremediation under high salinity conditions(Elsevier, 2019) Batista-García, Ramón; González-Abradeloa, Deborah; Pérez-Llano, Yordanis; Peidro-Guzmán, Heidy; Sánchez-Carbente, María; Folch, Jorge Luis; Aranda-Ballesteros, Elizabet; Vaidyanathan, Vinoth Kumar; Cabana, Hubert; Gunde-Cimerman, NinaPolycyclic aromatic hydrocarbons (PAH) and pharmaceutical compounds (PhC) are xenobiotics present in many saline wastewaters. Although fungi are known for their ability to remove xenobiotics, the potential of halophilic fungi to degrade highly persistent pollutants was not yet investigated. The use of two halophilic fungi, Aspergillus sydowii and Aspergillus destruens, for the elimination of PAH and PhC at saline conditions was studied. In saline synthetic medium both fungi used benzo-α-pyrene and phenanthrene as sole carbon source and removed over 90% of both PAH, A. sydowii due to biodegradation and A. destruens to bioadsorption. They removed 100% of a mixture of fifteen PAH in saline biorefinery wastewater. Test using Cucumis sativus demonstrated that wastewater treated with the two fungi lowered considerably the phytotoxicity. This study is the first demonstration that ascomycetous halophilic fungi, in contrast to other fungi (and in particular basidiomycetes) can be used for mycotreatments under salinity conditions.Ítem From lignocellulosic metagenomes to lignocellulolytic genes: trends, challenges and future prospects(Wiley, 2016) Batista-García, Ramón; Sánchez-Carbente, María; Talia, Paola; Jackson, Stephen A.; O'Leary, Niall D.; Dobson, Alan D.W.; Folch, Jorge LuisLignocellulose is the most abundant biomass on Earth with immense potential to act as a primary resource for the production of a range of compounds currently obtained from fossil fuel sources. However, lignocellulosic feedstocks remain largely underexploited due to the complex mixture of recalcitrant polymers present, whose structural features hinder access to the utilizable monosaccharide reservoir within cellulose. Various fungi and bacteria have been identifi ed that can enzymatically decompose lignocellulose to its monomeric compounds for use as carbon sources. The investigation of such lignocellulolytic organisms has proven very useful in gaining primary insights into degradation processes and key microbial enzymes, but the established limitations of culture-based approaches suggest that we have yet to understand the full range of lignocellulolytic mechanisms, likely expressed within natural systems. In this review, we focus on metagenomic approaches to study lignocellulose degradation from structural and functional perspectives, which may provide novel insights into this process in order to rationally design methods for the extraction of compounds from biomass that could enhance biorefinery efficiencies.Ítem A Novel Expansin Protein from the White-Rot Fungus Schizophyllum commune(PLOS, 2015) Batista-García, Ramón; Tovar-Herrera, Omar; Sánchez-Carbente, María; Iracheta, María Magdalena; Arévalo-Niño, Katiushka; Folch, Jorge LuisA novel expansin protein (ScExlx1) was found, cloned and expressed from the Basidiomycete fungus Schizophylum commune. This protein showed the canonical features of plant expansins. ScExlx1 showed the ability to form “bubbles” in cotton fibers, reduce the size of avicel particles and enhance reducing sugar liberation from cotton fibers pretreated with the protein and then treated with cellulases. ScExlx1 was able to bind cellulose, birchwood xylan and chitin and this property was not affected by different sodium chloride concentrations. A novel property of ScExlx1 is its capacity to enhance reducing sugars (N-acetyl glucosamine) liberation from pretreated chitin and further added with chitinase, which has not been reported for any expansin or expansin-like protein. To the best of our knowledge, this is the first report of a bona fide fungal expansin found in a basidiomycete and we could express the bioactive protein in Pichia pastoris.Ítem A novel TctA citrate transporter from an activated sludge metagenome: Structural and mechanistic predictions for the TTT family(2014) Batista-García, Ramón; Sánchez-Reyes, Ayixon; Millán, César; González-Zuñiga, Víctor Manuel; Folch, Jorge Luis; Juárez-Ramírez, SoledadWe isolated a putative citrate transporter of the tripartite tricarboxylate transporter (TTT) class from a metagenomic library of activated sludge from a sewage treatment plant. The transporter, dubbed TctA_ar, shares ~50% sequence identity with TctA of Comamonas testosteroni (TctA_ct) and other b-Proteobacteria, and contains two 20-amino acid repeat signature sequences, considered a hallmark of this particular transporter class. The structures for both TctA_ar and TctA_ct were modeled with I-TASSER and two possible structures for this transporter family were proposed. Docking assays with citrate resulted in the corresponding sets of proposed critical residues for function. These models suggest functions for the 20-amino acid repeats in the context of the two different architectures. This constitutes the first attempt at structure modeling of the TTT family, to the best of our knowledge, and could aid functional understanding of this little-studied family.Ítem Identification of a novel carbohydrate esterase from Bjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling(Wiley, 2015-01) Batista-García, Ramón; Valdés, Gilberto; Cuervo, Laura; Sánchez-Carbente, María; Balcázar, Edgar; Lira, Verónica; Folch, Jorge LuisA new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg21 protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.Ítem Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride(PLOS, 2016) Batista-García, Ramón; Balcázar, Edgar; Méndez-Lorenzo, Luz Helena; Esquivel, Ulises; Savary, Oliver; Cabana, Hubert; Herrera-Estrella, Alfredo; Folch, Jorge Luis; Kumar, Vaidyanathan Vinoth; Ayala-Aceves, MarcelaFungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.Ítem Osmolyte Signatures for the Protection of Aspergillus sydowii Cells under Halophilic Conditions and Osmotic Shock(MDPI, 2021) Perez-Llano, YordanisÍtem Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions(John Wiley & Sons Ltd, 2021) Perez-Llano, YordanisÍtem Stress reshapes the physiological response of halophile fungi to salinity(MDPI, 2020-02) Perez-Llano, YordanisÍtem Haloadaptative responses of Aspergillus sydowii to extreme water deprivation: morphology, compatible solutes, and oxidative stress at NaCl saturation(MDPI, 2020-11) Perez-Llano, YordanisÍtem First demonstration that ascomycetous halophilic fungi (Aspergillus sydowii and Aspergillus destruens) are useful in xenobiotic mycoremediation under high salinity conditions(Elsevier, 2019-02-02) Perez-Llano, Yordanis