Production of the Tremorgenic Mycotoxins Verruculogen and Fumitremorgin B by Penicillium piscarium Westling

The tremorgenic mycotoxins verruculogen and fumitremorgin B were isolated from Penicillium piscarium Westling. The coexistence of these tremorgens in culture has previously been reported for one other unrelated fungal species, Aspergillus caespitosus Raper and Thom, and lends further support to the suggestion that the tremorgens have a common biosynthetic origin.     

Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC–MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus.     

Identification of csypyrone B2 and B3 as the minor products of Aspergillus oryzae type III polyketide synthase CsyB

Since our first report on the identification of the fungal type III polyketide synthase (PKS) genes csyA～D in Aspergillus oryzae RIB40, type III PKS homologues have also been found in other fungal species. We previously reported the isolation and structural determination of csypyrone B1 as the main product of CsyB when inductively expressed in Aspergillus oryzae. Herein we report the isolation and identification of the two minor products of the csyB transformant in addition to csypyrone B1 as 4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid and 5-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)pentanoic acid. These compounds were named csypyrone B2 and B3, respectively, and both are homologues of main product csypyrone B1 with different side chain lengths. This result suggests that the carbon skeleton of the csypyrone B precursor is constructed by the condensation of fatty acyl-CoA and acetylmalonyl-CoA followed by pyrone formation. The alkyl side chain of the precursor may be oxidatively cleaved by enzyme(s) in the host fungus to give variations of csypyrone B with propanoic acid, butyric acid, or pentanoic acid side chains.     

异戊烯基化吲哚类生物碱的化学酶合成

异戊烯基化吲哚类生物碱广泛存在于麦角菌、青霉菌和曲霉菌中,具有一定的药理学活性,与未异戊烯基化的前体在生物活性方面具有明显的差异.曲霉菌中的某些异戊烯基化吲哚类生物碱具有抗癌活性,如烟曲霉毒素C（fumitremorgin C）、tryprostatin B,但其天然产量低且不易分离,利用化学酶合成法可很容易地将前体转化为异戊烯基化吲哚类生物碱.异戊烯基转移酶FtmPT1对二甲丙烯基二磷酸（dimethylallyl diphosphate,DMAPP）具有专一性,但可以接受不同的芳香族底物.早期研究发现,FtmPT1能接受含色氨酸的不同环二肽为底物,但以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物时,酶的相对活性很低,其产物量少,无法用于合成产物.本实验通过优化酶反应条件来提高其产量.将已构建的含ftmPT1的质粒在大肠杆菌中诱导表达,经Ni-NTA亲和柱纯化后用于酶反应.实验结果表明,通过增加酶量（终浓度2.8 μmol/L）、延长培养时间（37 ℃,24 h）,以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物的酶反应产率分别达到49.3%和21.3%,产物经¹H^-NMR、¹H-¹H-COSY和ESI-MS鉴定,其结果与预期吻合.据检索,这2个化合物均为新化合物,分别命名为cyclo-C2-1′-DMA-L-Trp-L-Tyr和cyclo-C2-1′-DMA-L-Trp-L-Phe.     

Study of codon bias perspective of fungal xylanase gene by multivariate analysis

Fungal xylanases has important applications in food, baking, pulp and paper industries in addition to various other industries. Xylanases are produced extensively by both bacterial and fungal sources and has tremendous potential of being active at extremes of temperature and pH. In the present study an effort has been made to explore the codon bias perspective of this potential enzyme using bioinformatics tools. Multivariate analysis has been used as a tool to study codon bias perspectives of xylanases. It was further observed that the codon usage of xylanases genes from different fungal sources is not similar and to reveal this phenomenon the relative synonymous codon usage (RSCU) and base composition variation in fungal xylanase genes were also studied. The codon biasing data like GC content at third position (GC_3S), effective codon number (N_C), codon adaptive index (CAI) were further analyzed with statistical softwares like Sigma1plot 9.0 and Systat 11.0. Furthermore, study of translation selection was also performed to verify the influences of codon usage variation among the 94 xylanase genes. In the present study xylanase gene from 12 organisms were analyzed and codon usages of all xylanases from each organism were compared separately. Analysis indicates biased codon among all 12 fungi taken for study with Aspergillus nidulans, Chaetomium globosum, Aspergillus terreus and Aspergillus clavatus showing maximum biasing. N_C plot and correspondence analysis on relative synonymous codon usage indicate that mutation bias and translation selection influences codon usage variation in fungal xylanase gene. To reveal the relative synonymous codon usage and base composition variation in xylanase, 94 genes from 12 fungi were used as model system.     

The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus

Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.     

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development,Aflatoxin Accumulation,and Seed Infection

Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Discovery of a novel Aurora B inhibitor GSK650394 with potent anticancer and anti-aspergillus fumigatus dual efficacies in vitro

Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus, with IC₅₀ values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus. Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Mycoremediation of crude oil contaminated soil by specific fungi isolated from Dhahran in Saudi Arabia

Crude oil biodegrading microorganism considers the key role for environmental preserving. In this investigation, crude oil biodegrading fungal strains have been isolated in polluted soil of crude-oil at khurais oil ground in Kingdom of Saudi Arabia. Among of 22 fungal isolates, only three isolates reflected potential capability for oil degradation. These isolates were identified and submitted to GenBank as (A1) Aspergillus polyporicola (MT448790), (A2) Aspergillus spelaeus (MT448791) and (A3) Aspergillus niger (MT459302) through internal-transcribed spacer-regions (ITS1&ITS2) for sequencing in molecular marker. Comparing with controls, strain (A1) Aspergillus niger was superior for biodegradation ability (58%) comparing with Aspergillus polyporicola and Aspergillus spelaeus degrading were showed 47 and 51% respectively. Employed CO₂ evolution as indicator for petroleum oil biodegradation by the fungal isolates reflected that, Aspergillus niger emission highest CO2 (28.6%) comparing with Aspergillus spelaeus and Aspergillus polyporicola which showed 13% and 12.4% respectively. capability of Aspergillus sp. to tolerate and adapted oil pollutants with successful growth rate on them, indicated that it can be employed as mycoremediation agent for recovering restoring ecosystem when contaminated by crude oil.     

The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.     

Fungal Air Spora at Ibadan, Nigeria

The fungal air spora at Ibadan, Nigeria, was investigated by using Casella Slit Samplers. Three sites, incorporating three locations at each site, were selected for the exposure of replicate plates during sampling. To provide data on a wide range of saprophytic and pathogenic fungal spores, isolations were made on Sabouraud dextrose agar and malt agar plates incubated at 26 and 37 C. Altogether over 60,000 fungal colonies were isolated and counted during the 12-month sampling period. The prevalent fungal genera recorded were: Cladosporum, Curvularia, Fusarium, Aspergillus, Penicillium, Pithomyces, Aureobasidium, Geotrichum, Phoma, Nigrospora, Epicoccum, and Neurospora. The wet and dry seasons (indicated by the temperature, relative humidity, and rainfall data) caused seasonal periodicity in colony numbers. The influence of culture media on the isolated colonies was not significant when the total number of isolated colonies were considered on a monthly basis, but in reviewing a few of the fungal genera there were marked differences between the two media, especially with Pithomyces. Attempts were made to identify some of the isolated colonies by species, e.g., Aspergillus carneus, Aspergillus flavus, Aspergillus fumigatus, Curvularia geniculata, Fusarium oxysporum, Penicillium herquei, Pithomyces chartaum, Rhizopus arrhizus, and Syncephalastrum racemosum. Such identifications provide a basis for further studies on the role of these fungal species in the frontier problem of contamination and biodegradation of drugs and pharmaceuticals, allergies and other problems in the local environment.     

Unveiling the Functions of the VosA-VelB Target Gene vidD in Aspergillus nidulans

The velvet regulators VosA and VelB are primarily involved in spore maturation and dormancy. Previous studies found that the VosA-VelB hetero-complex coordinates certain target genes that are related to fungal differentiation and conidial maturation in Aspergillus nidulans. Here, we characterized the VosA/VelB-inhibited developmental gene vidD in A. nidulans. Phenotypic analyses demonstrated that the vidD deleted mutant exhibited defect fungal growth, a reduced number of conidia, and delayed formation of sexual fruiting bodies. The deletion of vidD decreased the amount of conidial trehalose, increased the sensitivity against heat stress, and reduced the conidial viability. Moreover, the absence of vidD resulted in increased production of sterigmatocystin. Together, these results show that VidD is required for proper fungal growth, development, and sterigmatocystin production in A. nidulans.     

A Novel Motif in Fungal Class 1 Histone Deacetylases Is Essential for Growth and Development of Aspergillus

Acetylation of the N-terminal tails of core histones is an important regulatory mechanism in eukaryotic organisms. In filamentous fungi, little is known about the enzymes that modify histone tails. However, it is increasingly evident that histone deacetylases and histone acetyltransferases are critical factors for the regulation of genes involved in fungal pathogenicity, stress response, and production of secondary metabolites such as antibiotics or fungal toxins. Here, we show that depletion of RpdA, an RPD3-type histone deacetylase of Aspergillus nidulans, leads to a pronounced reduction of growth and sporulation of the fungus. We demonstrate that a so far unnoticed motif in the C terminus of fungal RpdA histone deacetylases is required for the catalytic activity of the enzyme and consequently is essential for the viability of A. nidulans. Moreover, we provide evidence that this motif is also crucial for the survival of other, if not all, filamentous fungi, including pathogens such as Aspergillus fumigatus or Cochliobolus carbonum. Thus, the extended C terminus of RpdA-type enzymes represents a promising target for fungal-specific histone deacetylase-inhibitors that may have potential as novel antifungal compounds with medical and agricultural applications.