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Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans  [PDF]
Katy L. Ryan,Christopher T. Moore,Daniel G. Panaccione
Toxins , 2013, DOI: 10.3390/toxins5020445
Abstract: Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes, dmaW, easF, easE, and easC, are necessary for pathway steps prior to chanoclavine-I; however, the sufficiency of these genes for chanoclavine-I synthesis has not been established. A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillus fumigatus and transformed into Aspergillus nidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillus nidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis.
Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach  [PDF]
Morten Thrane Nielsen, Jakob Bl？sbjerg Nielsen, Dianna Chinyere Anyaogu, Dorte Koefoed Holm, Kristian Fog Nielsen, Thomas Ostenfeld Larsen, Uffe Hasbro Mortensen
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072871
Abstract: Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.
Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans  [PDF]
Natarajaswamy Kalleda, Aruna Naorem, Rajam V. Manchikatla
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075443
Abstract: Background Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. Methodology/Principal Findings Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. Conclusions/Significance We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.
The nucleation of microtubules in Aspergillus nidulans germlings
Andrade-Monteiro, Cristina de;Martinez-Rossi, Nilce M.;
Genetics and Molecular Biology , 1999, DOI: 10.1590/S1415-47571999000300004
Abstract: microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. in fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. we used the antimicrotubule drug benomyl in block/release experiments to depolymerize and repolymerize microtubules in aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. twenty seconds after release from benomyl short microtubules were formed from several bright (immunofluorescent) dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in a. nidulans germlings. since nuclear movement is dependent on microtubules in a. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants) have some obvious microtubule defect. cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. however, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. this suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.
The nucleation of microtubules in Aspergillus nidulans germlings  [cached]
Andrade-Monteiro Cristina de,Martinez-Rossi Nilce M.
Genetics and Molecular Biology , 1999,
Abstract: Microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. In fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. We used the antimicrotubule drug Benomyl in block/release experiments to depolymerize and repolymerize microtubules in Aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. Twenty seconds after release from Benomyl short microtubules were formed from several bright (immunofluorescent) dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in A. nidulans germlings. Since nuclear movement is dependent on microtubules in A. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants) have some obvious microtubule defect. Cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. However, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. This suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.
Analysis of Aspergillus nidulans metabolism at the genome-scale
Helga David, ？lknur ？ ？z？elik, Gerald Hofmann, Jens Nielsen
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-163
Abstract: In this work, we have manually assigned functions to 472 orphan genes in the metabolism of A. nidulans, by using a pathway-driven approach and by employing comparative genomics tools based on sequence similarity. The central metabolism of A. nidulans, as well as biosynthetic pathways of relevant secondary metabolites, was reconstructed based on detailed metabolic reconstructions available for A. niger and Saccharomyces cerevisiae, and information on the genetics, biochemistry and physiology of A. nidulans. Thereby, it was possible to identify metabolic functions without a gene associated, and to look for candidate ORFs in the genome of A. nidulans by comparing its sequence to sequences of well-characterized genes in other species encoding the function of interest. A classification system, based on defined criteria, was developed for evaluating and selecting the ORFs among the candidates, in an objective and systematic manner. The functional assignments served as a basis to develop a mathematical model, linking 666 genes (both previously and newly annotated) to metabolic roles. The model was used to simulate metabolic behavior and additionally to integrate, analyze and interpret large-scale gene expression data concerning a study on glucose repression, thereby providing a means of upgrading the information content of experimental data and getting further insight into this phenomenon in A. nidulans.We demonstrate how pathway modeling of A. nidulans can be used as an approach to improve the functional annotation of the genome of this organism. Furthermore we show how the metabolic model establishes functional links between genes, enabling the upgrade of the information content of transcriptome data.Aspergillus nidulans, also known as Emericella nidulans, as it can undergo sexual reproduction in its life cycle in addition to the non-perfect (asexually reproducing) form that characterizes aspergilli, is an important member of the filamentous fungal genus Aspergillus. Thi
Identification of Glucose Transporters in Aspergillus nidulans  [PDF]
Thaila Fernanda dos Reis, Jo？o Filipe Menino, Vinícius Leite Pedro Bom, Neil Andrew Brown, Ana Cristina Colabardini, Marcela Savoldi, Maria Helena S. Goldman, Fernando Rodrigues, Gustavo Henrique Goldman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081412
Abstract: To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
Genotoxic evaluation of sodium nitroprusside in Aspergillus nidulans
Chiuchetta, Simone Jurema Ruggeri;Castro-Prado, Marialba Avezum Alves de;
Genetics and Molecular Biology , 2005, DOI: 10.1590/S1415-47572005000500023
Abstract: the exogenous nitric oxide donor, sodium nitroprusside, evaluated the recombinogenic potential of nitric oxide. drug inhibited mycelial growth and conidiation in a757 aspergillus nidulans master strain. two heterozygous diploid strains, one wild (uvsh+//uvsh+) and the other defective to dna repair (uvsh//uvsh) were used for recombinagenesis tests. sodium nitroprusside recombinogenic effect was evaluated by the induction of homozygosis of recessive genes, originally present in heterozygous condition. results show that sodium nitroprusside (40 mm, 80 mm and 160 mm) is effective in inducing mitotic crossing-over in diploid cells of a. nidulans.
Genetics of Polyketide Metabolism in Aspergillus nidulans  [PDF]
Marie L. Klejnstrup,Rasmus J. N. Frandsen,Dorte K. Holm,Morten T. Nielsen,Uffe H. Mortensen,Thomas O. Larsen,Jakob B. Nielsen
Metabolites , 2012, DOI: 10.3390/metabo2010100
Abstract: Secondary metabolites are small molecules that show large structural diversity and a broad range of bioactivities. Some metabolites are attractive as drugs or pigments while others act as harmful mycotoxins. Filamentous fungi have the capacity to produce a wide array of secondary metabolites including polyketides. The majority of genes required for production of these metabolites are mostly organized in gene clusters, which often are silent or barely expressed under laboratory conditions, making discovery and analysis difficult. Fortunately, the genome sequences of several filamentous fungi are publicly available, greatly facilitating the establishment of links between genes and metabolites. This review covers the attempts being made to trigger the activation of polyketide metabolism in the fungal model organism Aspergillus nidulans. Moreover, it will provide an overview of the pathways where ten polyketide synthase genes have been coupled to polyketide products. Therefore, the proposed biosynthesis of the following metabolites will be presented; naphthopyrone, sterigmatocystin, aspyridones, emericellamides, asperthecin, asperfuranone, monodictyphenone/emodin, orsellinic acid, and the austinols.
The Putative C2H2 Transcription Factor MtfA Is a Novel Regulator of Secondary Metabolism and Morphogenesis in Aspergillus nidulans  [PDF]
Vellaisamy Ramamoorthy, Sourabh Dhingra, Alexander Kincaid, Sourabha Shantappa, Xuehuan Feng, Ana M. Calvo
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074122
Abstract: Secondary metabolism in the model fungus Aspergillus nidulans is controlled by the conserved global regulator VeA, which also governs morphological differentiation. Among the secondary metabolites regulated by VeA is the mycotoxin sterigmatocystin (ST). The presence of VeA is necessary for the biosynthesis of this carcinogenic compound. We identified a revertant mutant able to synthesize ST intermediates in the absence of VeA. The point mutation occurred at the coding region of a gene encoding a novel putative C2H2 zinc finger domain transcription factor that we denominated mtfA. The A. nidulans mtfA gene product localizes at nuclei independently of the illumination regime. Deletion of the mtfA gene restores mycotoxin biosynthesis in the absence of veA, but drastically reduced mycotoxin production when mtfA gene expression was altered, by deletion or overexpression, in A. nidulans strains with a veA wild-type allele. Our study revealed that mtfA regulates ST production by affecting the expression of the specific ST gene cluster activator aflR. Importantly, mtfA is also a regulator of other secondary metabolism gene clusters, such as genes responsible for the synthesis of terrequinone and penicillin. As in the case of ST, deletion or overexpression of mtfA was also detrimental for the expression of terrequinone genes. Deletion of mtfA also decreased the expression of the genes in the penicillin gene cluster, reducing penicillin production. However, in this case, over-expression of mtfA enhanced the transcription of penicillin genes, increasing penicillin production more than 5 fold with respect to the control. Importantly, in addition to its effect on secondary metabolism, mtfA also affects asexual and sexual development in A. nidulans. Deletion of mtfA results in a reduction of conidiation and sexual stage. We found mtfA putative orthologs conserved in other fungal species.
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