Publications by authors named "Jae-Hyuk Yu"

120 Publications

DUG pathway governs degradation of intracellular glutathione in .

Appl Environ Microbiol 2021 Feb 26. Epub 2021 Feb 26.

Department of Molecular Biotechnology and Microbiology, Faculty of Sciences and Technology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.

Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance to deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigate functions of the and genes in the model fungus These genes are orthologues of involved in cytosolic GSH degradation in the yeast.The deletion (Δ) of , , or both resulted in a moderate increase in the GSH content in the mycelia grown on glucose, reduced conidia production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding MAP kinase pathway elements () or regulatory proteins of conidiogenesis and sexual differentiation (, ) were down-regulated in the mutant.Deletion of and/or slowed down the depletion of GSH pools under carbon starvation. It also reduced accumulation of reactive oxygen species, decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions - in contrast to mycotoxin production - were controlled at post-transcriptional level. We suggest that GSH connects starvation and redox regulation to each other: Cells utilize GSH as stored carbon source during starvation. The reduction of GSH content alters the redox state activating regulatory pathways responsible for carbon starvation stress responses.Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, secondary metabolite production (including mycotoxin formation) as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity or oxidative stress tolerance of a fungal strain is manipulated.
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http://dx.doi.org/10.1128/AEM.01321-20DOI Listing
February 2021

Transcriptomic, Protein-DNA Interaction, and Metabolomic Studies of VosA, VelB, and WetA in Aspergillus nidulans Asexual Spores.

mBio 2021 02 9;12(1). Epub 2021 Feb 9.

School of Food Science and Biotechnology, Kyungpook National University, Daegu, Republic of Korea

In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination. Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other species such as the major human pathogen and the aflatoxin producer .
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http://dx.doi.org/10.1128/mBio.03128-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885118PMC
February 2021

Analysis of E.U. Rapid Alert System (RASFF) Notifications for Aflatoxins in Exported U.S. Food and Feed Products for 2010-2019.

Toxins (Basel) 2021 Jan 26;13(2). Epub 2021 Jan 26.

Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.

The most common, toxic, and carcinogenic mycotoxins found in human food and animal feed are the aflatoxins (AFs). The United States is a leading exporter of various nuts, with a marketing value of $9.1 billion in 2019; the European Union countries are the major importers of U.S. nuts. In the past few years, border rejections and notifications for U.S. tree nuts and peanuts exported to the E.U. countries have increased due to AF contamination. In this work, we analyzed notifications from the "Rapid Alert System for Food and Feed (RASFF)" on U.S. food and feed products contaminated with mycotoxins, primarily AFs, for the 10-year period 2010-2019. Almost 95% of U.S. mycotoxin RASFF notifications were reported for foods and only 5% for feeds. We found that 98.9% of the U.S. food notifications on mycotoxins were due to the AF contamination in almond, peanut, and pistachio nuts. Over half of these notifications (57.9%) were due to total AF levels greater than the FDA action level in food of 20 ng g. The Netherlands issued 27% of the AF notifications for U.S. nuts. Border rejection was reported for more than 78% of AF notifications in U.S. nuts. All U.S. feed notifications on mycotoxins occurred due to the AF contamination. Our research contributes to better understanding the main reasons behind RASFF mycotoxins notifications of U.S. food and feed products destined to E.U. countries. Furthermore, we speculate possible causes of this problem and provide a potential solution that could minimize the number of notifications for U.S. agricultural export market.
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http://dx.doi.org/10.3390/toxins13020090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910969PMC
January 2021

Development of a novel homogeneous immunoassay using the engineered luminescent enzyme NanoLuc for the quantification of the mycotoxin fumonisin B1.

Biosens Bioelectron 2021 Apr 30;177:112939. Epub 2020 Dec 30.

Department of Bacteriology, Food Research Institute, University of Wisconsin-Madison, Madison, WI, 53706, USA; Department of Systems Biotechnology, Konkuk University, Seoul, Korea. Electronic address:

Compared to the traditional heterogeneous assays, a homogeneous immunoassay is a preferred format for its simplicity. By cloning and isolating luminescent proteins from bioluminescent organisms, bioluminescence has been widely used for various biological applications. In this study, we present the development of a homogeneous luminescence immunoassay (FNanoBiT assay) for detection of fumonisin B1 (FB1), based on the binding of two subunits of an engineered luminescent protein (NanoLuc). For the detection of the mycotoxin FB1 in foods, the anti-fumonisin antibody was conjugated to the large subunit of NanoLuc (FLgBiT), and the FB1 was conjugated to the small subunit (FSmBiT). The conjugates were used for the detection of FB1 in a competitive immunoassay format without the need of a secondary antibody, or washing steps. The developed FNanoBiT assay revealed high specificity toward FB1 with no cross-reactivity with other mycotoxins, and it demonstrated acceptable recovery (higher than 94%) and relative standard deviation from spiked maize samples. Further, the assay was successfully applied for the detection of FB1 in naturally contaminated maize, with a dynamic range of 0.533-6.81 ng mL-1 and a detection limit of 0.079 ng mL-1. The results derived with FNanoBiT assay of all spiked samples showed a strong correlation to those obtained by the High-performance liquid chromatography method. Thus, the FNanoBiT based homogeneous immunoassay could be used as a rapid, and simple tool for the analysis of mycotoxin-contaminated foods.
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http://dx.doi.org/10.1016/j.bios.2020.112939DOI Listing
April 2021

Complete mitochondrial genome sequence of Afla-Guard, commercially available non-toxigenic .

Mitochondrial DNA B Resour 2020 Oct 21;5(3):3590-3592. Epub 2020 Oct 21.

Department of Pharmaceutical Engineering, Woosuk University, Wanju, Republic of Korea.

Afla-Guard is a commercial non-toxigenic strain used to decrease aflatoxin contamination level in field. Its mitochondrial genome was sequenced, showing that its length is 29,208 bp with typical configuration of mitochondrial genome. 17 SNPs and 27 INDELs were identified by comparing with previous mitochondrial genome. Phylogenetic trees present that of Afla-Guard was clustered with the previous mitochondrial genome.
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http://dx.doi.org/10.1080/23802359.2020.1825129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594763PMC
October 2020

Complete mitochondrial genome sequence of SRRC1009: insight of intraspecific variations on mitochondrial genomes.

Mitochondrial DNA B Resour 2020 Oct 21;5(3):3585-3587. Epub 2020 Oct 21.

Department of Pharmaceutical Engineering, Woosuk University, Wanju, Republic of Korea.

The mitogenome of SRRC1009 was sequenced to investigate intraspecific variations on mitochondrial genomes of . It shows 29,202 bp with a typical configuration of mitogenome. Sixteen SNPs and 22 INDELs and 17 SNPs and 27 INDELs were identified against AflaGuard and JQ355000, respectively. Phylogenetic trees present in the three mitochondrial genomes were clustered with mitochondrial genome in one clade.
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http://dx.doi.org/10.1080/23802359.2020.1771226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594760PMC
October 2020

Revised Transcriptome-Based Gene Annotation for Aspergillus flavus Strain NRRL 3357.

Microbiol Resour Announc 2020 Dec 3;9(49). Epub 2020 Dec 3.

Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA

is an agriculturally and medically important filamentous fungus that produces mycotoxins, including aflatoxins, which are potent carcinogens. Here, we generated short- and long-read transcript sequence data from the growth of strain NRRL 3357 under both typical and stress conditions to produce a new annotation of its genome.
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http://dx.doi.org/10.1128/MRA.01155-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714855PMC
December 2020

The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus.

mSphere 2020 11 11;5(6). Epub 2020 Nov 11.

Department of Microbiology, Graduate School, Daejeon University, Daejeon, Republic of Korea

The APSES transcription factor (TF) in species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus The deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators , , and were decreased in the Δ mutant compared to those in the wild type (WT). Moreover, Δ resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the Δ mutant. Comparative transcriptomic analyses revealed that the Δ mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the Δ mutant exhibited elevated production of GT, Δ strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the Δ mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.
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http://dx.doi.org/10.1128/mSphere.00998-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657592PMC
November 2020

Heterotrimeric G-protein signalers and RGSs in .

Pathogens 2020 Oct 28;9(11). Epub 2020 Oct 28.

Department of Microbiology, Graduate School, Daejeon University, Daejeon 34520, Korea.

The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus . In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in .
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http://dx.doi.org/10.3390/pathogens9110902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693823PMC
October 2020

Velvet activated McrA plays a key role in cellular and metabolic development in Aspergillus nidulans.

Sci Rep 2020 09 15;10(1):15075. Epub 2020 Sep 15.

Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, 53706, USA.

McrA is a key transcription factor that functions as a global repressor of fungal secondary metabolism in Aspergillus species. Here, we report that mcrA is one of the VosA-VelB target genes and McrA governs the cellular and metabolic development in Aspergillus nidulans. The deletion of mcrA resulted in a reduced number of conidia and decreased mRNA levels of brlA, the key asexual developmental activator. In addition, the absence of mcrA led to a loss of long-term viability of asexual spores (conidia), which is likely associated with the lack of conidial trehalose and increased β-(1,3)-glucan levels in conidia. In supporting its repressive role, the mcrA deletion mutant conidia contain more amounts of sterigmatocystin and an unknown metabolite than the wild type conidia. While overexpression of mcrA caused the fluffy-autolytic phenotype coupled with accelerated cell death, deletion of mcrA did not fully suppress the developmental defects caused by the lack of the regulator of G-protein signaling protein FlbA. On the contrary to the cellular development, sterigmatocystin production was restored in the ΔflbA ΔmcrA double mutant, and overexpression of mcrA completely blocked the production of sterigmatocystin. Overall, McrA plays a multiple role in governing growth, development, spore viability, and secondary metabolism in A. nidulans.
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http://dx.doi.org/10.1038/s41598-020-72224-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493923PMC
September 2020

Comparative Analysis, Structural Insights, and Substrate/Drug Interaction of CYP128A1 in .

Int J Mol Sci 2020 Jul 8;21(14). Epub 2020 Jul 8.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa.

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms' primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different approaches to understand CYP128 P450 family's distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with , or together with and . The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in .
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http://dx.doi.org/10.3390/ijms21144816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404182PMC
July 2020

More P450s Are Involved in Secondary Metabolite Biosynthesis in Compared to , , and .

Int J Mol Sci 2020 Jul 7;21(13). Epub 2020 Jul 7.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa.

Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in and species, indicating its key role in the synthesis of secondary metabolites. species had a higher number of P450s than and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in species than in , mycobacterial, and cyanobacterial species, corroborating the superior capacity of species for generating diverse secondary metabolites. Functional analysis data mining confirmed that many P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of species, revealing the P450s' association with secondary metabolite synthesis in species. Future studies should include the selection of species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.
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http://dx.doi.org/10.3390/ijms21134814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369989PMC
July 2020

Increased Cd biosorption capability of Aspergillus nidulans elicited by crpA deletion.

J Basic Microbiol 2020 Jul 25;60(7):574-584. Epub 2020 May 25.

Department of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.

The P-type ATPase CrpA is an important Cu /Cd pump in the Aspergilli, significantly contributing to the heavy metal stress tolerance of these ascomycetous fungi. As expected, the deletion of crpA resulted in Cu /Cd -sensitive phenotypes in Aspergillus nidulans on stress agar plates inoculated with conidia. Nevertheless, paradoxical growth stimulations were observed with the ΔcrpA strain in both standard Cu stress agar plate experiments and cellophane colony harvest (CCH) cultures, when exposed to Cd . These observations reflect efficient compensatory mechanisms for the loss of CrpA operating under these experimental conditions. It is remarkable that the ΔcrpA strain showed a 2.7 times higher Cd biosorption capacity in CCH cultures, which may facilitate the development of new, fungal biomass-based bioremediation technologies to extract harmful Cd ions from the environment. The nullification of crpA also significantly changed the spatial distribution of Cu and Cd in CCH cultures, as demonstrated by the combined particle-induced X-ray emission and scanning transmission ion microscopy technique. Most important, the centers of gravity for Cu and Cd accumulations of the ΔcrpA colonies shifted toward the older regions as compared with wild-type surface cultures.
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http://dx.doi.org/10.1002/jobm.202000112DOI Listing
July 2020

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

Curr Genet 2020 Jun 14;66(3):621-633. Epub 2020 Feb 14.

School of Food Science and Biotechnology, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu, 41566, Republic of Korea.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.
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http://dx.doi.org/10.1007/s00294-020-01058-yDOI Listing
June 2020

A Liquid Chromatographic Method for Rapid and Sensitive Analysis of Aflatoxins in Laboratory Fungal Cultures.

Toxins (Basel) 2020 01 30;12(2). Epub 2020 Jan 30.

Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.

Culture methods supplemented with high-performance liquid chromatography (HPLC) technique provide a rapid and simple tool for detecting levels of aflatoxins (AFs) produced by fungi. This study presents a robust method for simultaneous quantification of aflatoxin (AF) B1, B2, G1, and G2 levels in several fungal cultivation states: submerged shake culture, liquid slant culture, and solid-state culture. The recovery of the method was evaluated by spiking a mixture of AFs at several concentrations to the test medium. The applicability of the method was evaluated by using aflatoxigenic and non-aflatoxigenic . A HPLC coupled with the diode array (DAD) and fluorescence (FLD) detectors was used to determine the presence and amounts of AFs. Both detectors showed high sensitivity in detecting spiked AFs or AFs produced in situ by toxigenic fungi. Our methods showed 76%-88% recovery from medium spiked with 2.5, 10, 50, 100, and 500 ng/mL AFs. The limit of quantification (LOQ) for AFs were 2.5 to 5.0 ng/mL with DAD and 0.025 to 2.5 ng/mL with FLD. In this work, we described in detail a protocol, which can be considered the foremost and only verified method, to extract, detect, and quantify AFs employing both aflatoxigenic and non-toxigenic .
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http://dx.doi.org/10.3390/toxins12020093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076963PMC
January 2020

Nitric oxide as a developmental and metabolic signal in filamentous fungi.

Mol Microbiol 2020 05 6;113(5):872-882. Epub 2020 Feb 6.

Key Laboratory for Biotechnology of Medicinal Plants, Jiangsu Normal University, Xuzhou, China.

The short-lived hydrophobic gas nitric oxide (NO) is a broadly conserved signaling molecule in all domains of life, including the ubiquitous and versatile filamentous fungi (molds). Several studies have suggested that NO plays a vast and diverse signaling role in molds. In this review, we summarize NO-mediated signaling and the biosynthesis and degradation of NO in molds, and highlight the recent advances in understanding the NO-mediated regulation of morphological and physiological processes throughout the fungal life cycle. In particular, we describe the role of NO in molds as a signaling molecule that modulates asexual and sexual development, the formation of infection body appressorium, and the production of secondary metabolites (SMs). In addition, we also summarize NO detoxification and protective mechanisms against nitrooxidative stress.
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http://dx.doi.org/10.1111/mmi.14465DOI Listing
May 2020

Comprehensive Analyses of Cytochrome P450 Monooxygenases and Secondary Metabolite Biosynthetic Gene Clusters in .

Int J Mol Sci 2020 Jan 19;21(2). Epub 2020 Jan 19.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa.

The prokaryotic phylum are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria , and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.
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http://dx.doi.org/10.3390/ijms21020656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014017PMC
January 2020

The Regulator VosA Governs Survival and Secondary Metabolism of Sexual Spores in .

Genes (Basel) 2020 01 16;11(1). Epub 2020 Jan 16.

Department of Pharmaceutical Engineering, Woosuk University, Wanju 55338, Korea.

The regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus . In the present study, we characterize the roles of VosA in sexual spores (ascospores) in . . During ascospore maturation, the deletion of causes a rapid decrease in spore viability. The absence of also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of results in the downregulation of various genes involved in primary metabolism. In addition, deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in .
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http://dx.doi.org/10.3390/genes11010103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016683PMC
January 2020

Transcriptomic and Functional Studies of the RGS Protein Rax1 in Aspergillus fumigatus.

Pathogens 2019 Dec 31;9(1). Epub 2019 Dec 31.

Department of Microbiology, Graduate School, Daejeon University, Daejeon 34520, Korea.

In the comparative transcriptomic studies of wild type (WT) and rax1 null mutant strains, we obtained an average of 22,222,727 reads of 101 bp per sample and found that 183 genes showed greater than 2.0-fold differential expression, where 92 and 91 genes were up-and down-regulated in rax1 compared to WT, respectively. In accordance with the significantly reduced levels of gliM and casB transcripts in the absence of rax1, the rax1 mutant exhibited increased sensitivity to exogenous gliotoxin (GT) without affecting levels of GT production. Moreover, rax1 resulted in significantly restricted colony growth and reduced viability under endoplasmic reticulum stress condition. In summary, Rax1 positively affects expression of and metacaspase genes.
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http://dx.doi.org/10.3390/pathogens9010036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168642PMC
December 2019

RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen .

Int J Mol Sci 2019 Nov 11;20(22). Epub 2019 Nov 11.

Department of Microbiology, Graduate School, Daejeon University, Daejeon 34520, Korea.

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus , six RGS protein-encoding genes are present. To characterize the gene predicted to encode a protein with an RGS domain, we generated an null mutant and observed the phenotypes of the mutant. The deletion (Δ) of resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators , and are elevated in the Δ mutant. Moreover, Δ resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by Δ. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of , and the Δ spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the Δ mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by Δ and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation-likely by attenuating PKA signaling.
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http://dx.doi.org/10.3390/ijms20225628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888639PMC
November 2019

The velvet repressed vidA gene plays a key role in governing development in Aspergillus nidulans.

J Microbiol 2019 Oct 28;57(10):893-899. Epub 2019 Aug 28.

School of Food Science and Biotechnology, Institute of Agricultural Science and Technology, Kyungpook National University, Daegu, 41566, Republic of Korea.

Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a CH zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and β-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.
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http://dx.doi.org/10.1007/s12275-019-9214-4DOI Listing
October 2019

Effects of Different G-Protein α-Subunits on Growth, Development and Secondary Metabolism of M7.

Front Microbiol 2019 9;10:1555. Epub 2019 Jul 9.

Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China.

Strains of filamentous fungal species have been used to produce fermented foods in Asian countries, such as China, Japan, and The Korean Peninsula, for nearly 2,000 years. At present, their fermented products are widely used as food additives and nutraceutical supplements worldwide owing to their production of beneficial secondary metabolites. Heterotrimeric G-protein signaling pathways participate in regulating multiple biological processes in fungi. Previously, we identified three M7 G-protein α subunits (Mga1-3) and demonstrated that Mga1 can regulate growth, reproduction and some secondary metabolites' production. Here, we systematically analyzed and compared the roles of 1-3 by combining single- and double-gene(s) knockouts and their transcriptomic data. First, 2 and 3 knock-out mutants and pairwise combinations of 1-3 deletion strains were generated. Then the changes in growth, development and the main secondary metabolites, pigments and citrinin, in these mutants were systematically compared with M7. Moreover, RNA-Seq analyses of these mutants were performed. All three Gα subunits worked together to regulate biological processes in M7, with Mga1 playing a major role, while Mga2 and Mga3 playing supplemental roles. According to the existing literatures which we can find, gene knock-out mutants of the pairwise combination of 1-3 and their transcriptome analysis are first reported in this study. The current results have clearly demonstrated the functional division of Mga1-3 in M7, and could provide a deeper understanding of the effects of different Gα subunits on growth, development and secondary metabolism in other filamentous fungi.
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http://dx.doi.org/10.3389/fmicb.2019.01555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632705PMC
July 2019

Distribution and Diversity of Cytochrome P450 Monooxygenases in the Fungal Class .

Int J Mol Sci 2019 Jun 13;20(12). Epub 2019 Jun 13.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa.

, a fungal class in the subphylum , contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of , inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of . Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of .
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http://dx.doi.org/10.3390/ijms20122889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627453PMC
June 2019

Cytochrome P450 Monooxygenase CYP139 Family Involved in the Synthesis of Secondary Metabolites in 824 Mycobacterial Species.

Int J Mol Sci 2019 May 31;20(11). Epub 2019 May 31.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa.

Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, , is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: complex (850-species), complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily "A". CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts' metabolism and immune system.
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http://dx.doi.org/10.3390/ijms20112690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600245PMC
May 2019

Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[]Pyrene by Species.

mBio 2019 05 28;10(3). Epub 2019 May 28.

Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin, USA

Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several species are capable of degrading BaP. Exposing cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in We further demonstrate that the fungal NF-κB-type regulators VeA and VelB are required for proper expression of in response to nutrient limitation and BaP degradation in Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. We are increasingly exposed to environmental pollutants, including the carcinogen benzo[]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.
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http://dx.doi.org/10.1128/mBio.00558-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538779PMC
May 2019

Efficient Adsorption on Benzoyl and Stearoyl Cellulose to Remove Phenanthrene and Pyrene from Aqueous Solution.

Polymers (Basel) 2018 Sep 19;10(9). Epub 2018 Sep 19.

Department of Systems Biotechnology, Microbial Carbohydrate Resource Bank (MCRB), Center for Biotechnology Research in UBITA (CBRU), Konkuk University, Seoul 05029, Korea.

Benzoyl and stearoyl acid grafted cellulose were synthesized by a simple chemical grafting method. Using these as chemical adsorbents, polycyclic aromatic hydrocarbons (PAHs), like pyrene and phenanthrene, were effectively removed from aqueous solution. The structural and morphological properties of the synthesized adsorbents were determined through X-ray diffraction analysis (XRD), thermal gravimetric analysis (TGA), Fourier transform infrared (FT-IR), FE-SEM, and NMR analyses. Through this method, it was confirmed that benzoyl and stearoyl acid were successfully grafted onto the surface of cellulose. The 5 mg of stearoyl grafted cellulose (St⁻Cell) remove 96.94% pyrene and 97.61% phenanthrene as compared to unmodified cellulose, which adsorbed 1.46% pyrene and 2.99% phenanthrene from 0.08 ppm pyrene and 0.8 ppm phenanthrene aqueous solution, suggesting that those results show a very efficient adsorption performance as compared to the unmodified cellulose.
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http://dx.doi.org/10.3390/polym10091042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403814PMC
September 2018

Similarities, variations, and evolution of cytochrome P450s in Streptomyces versus Mycobacterium.

Sci Rep 2019 03 8;9(1):3962. Epub 2019 Mar 8.

Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa, 3886, KwaZulu-Natal, South Africa.

Cytochrome P450 monooxygenases (P450s) found in all domains of life are known for their catalytic versatility and stereo- and regio-specific activity. While the impact of lifestyle on P450 evolution was reported in many eukaryotes, this remains to be addressed in bacteria. In this report, Streptomyces and Mycobacterium, belonging to the phylum Actinobacteria, were studied owing to their contrasting lifestyles and impacts on human. Analyses of all P450s and those predicted to be associated with secondary metabolism have revealed that different lifestyles have affected the evolution of P450s in these bacterial genera. We have found that while species in both genera have essentially the same number of P450s in the genome, Streptomyces P450s are much more diverse than those of Mycobacterium. Moreover, despite both belonging to Actinobacteria, only 21 P450 families were common, and 123 and 56 families were found to be unique to Streptomyces and Mycobacterium, respectively. The presence of a large and diverse number of P450s in Streptomyces secondary metabolism contributes to antibiotic diversity, helping to secure the niche. Conversely, based on the currently available functional data, types of secondary metabolic pathways and associated P450s, mycobacterial P450s seem to play a role in utilization or synthesis of lipids.
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http://dx.doi.org/10.1038/s41598-019-40646-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408508PMC
March 2019

RgsD negatively controls development, toxigenesis, stress response, and virulence in Aspergillus fumigatus.

Sci Rep 2019 01 28;9(1):811. Epub 2019 Jan 28.

Department of Life Science, Daejeon University, Daejeon, 34520, Republic of Korea.

The regulator of G protein signaling (RGS) domain proteins generally attenuate heterotrimeric G protein signaling, thereby fine-tune the duration and strength of signal transduction. In this study, we characterize the functions of RgsD, one of the six RGS domain proteins present in the human pathogenic fungus Aspergillus fumigatus. The deletion (Δ) of rgsD results in enhanced asexual sporulation coupled with increased mRNA levels of key developmental activators. Moreover, ΔrgsD leads to increased spore tolerance to UV and oxidative stress, which might be associated with the enhanced expression of melanin biosynthetic genes and increased amount of melanin. Yeast two-hybrid assays reveal that RgsD can interact with the three Gα proteins GpaB, GanA, and GpaA, showing the highest interaction potential with GpaB. Importantly, the ΔrgsD mutant shows elevated expression of genes in the cAMP-dependent protein kinase A (PKA) pathway and PKA catalytic activity. The ΔrgsD mutant also display increased gliotoxin production and elevated virulence toward Galleria mellonella wax moth larvae. Transcriptomic analyses using RNA-seq reveal the expression changes associated with the diverse phenotypic outcomes caused by ΔrgsD. Collectively, we conclude that RgsD attenuates cAMP-PKA signaling pathway and negatively regulates asexual development, toxigenesis, melanin production, and virulence in A. fumigatus.
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http://dx.doi.org/10.1038/s41598-018-37124-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349852PMC
January 2019

Disturbance in biosynthesis of arachidonic acid impairs the sexual development of the onion blight pathogen Stemphylium eturmiunum.

Curr Genet 2019 Jun 16;65(3):759-771. Epub 2019 Jan 16.

Key Laboratory for Biology of Vegetable Diseases and Insect Pests of Shandong Province, Department of Plant Pathology, Shandong Agricultural University, Taian, 271018, Shandong, China.

The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.
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http://dx.doi.org/10.1007/s00294-019-00930-wDOI Listing
June 2019

Genome Sequence of the Extremely Acidophilic Fungus Acidomyces richmondensis FRIK2901.

Microbiol Resour Announc 2018 Oct 25;7(16). Epub 2018 Oct 25.

Department of Food Science, University of Massachusetts, Amherst, Massachusetts, USA.

Acidomyces richmondensis is an extremophilic fungal species found in warm, acidic, and metal-rich environments. To improve upon the existing reference genome, we used PacBio and Illumina sequencing to assemble a highly contiguous 29.3-Mb genome of A. richmondensis FRIK2901.
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http://dx.doi.org/10.1128/MRA.01314-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256575PMC
October 2018