scholarly journals Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case

2011 ◽  
Vol 77 (9) ◽  
pp. 3044-3051 ◽  
Author(s):  
Bjarne G. Hansen ◽  
Bo Salomonsen ◽  
Morten T. Nielsen ◽  
Jakob B. Nielsen ◽  
Niels B. Hansen ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Mycophenolic Acid ◽  
Polyketide Synthase ◽  
Test Case ◽  
Content Type ◽  
Genomic Location ◽  
Polyketide Synthase Gene ◽  
Penicillium Brevicompactum

ABSTRACTAssigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location ofAspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp largempaCgene fromPenicillium brevicompactumtoA. nidulans. In parallel, we produced defined mutant derivatives ofmpaC, and the combined analysis ofA. nidulansstrains expressingmpaCor mutatedmpaCgenes unequivocally demonstrated thatmpaCindeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.

scholarly journals Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

2011 ◽  
Vol 77 (9) ◽  
pp. 3035-3043 ◽  
Author(s):  
Torsten Bak Regueira ◽  
Kanchana Rueksomtawin Kildegaard ◽  
Bjarne Gram Hansen ◽  
Uffe H. Mortensen ◽  
Christian Hertweck ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mycophenolic Acid ◽  
Molecular Basis ◽  
Polyketide Synthase ◽  
Functional Characterization ◽  
Gene Encoding ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Polyketide Synthase Gene ◽  
Penicillium Brevicompactum

ABSTRACTMycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production inPenicillium brevicompactum. mpaCresides in what most likely is a 25-kb gene cluster in the genome ofPenicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase genempaCofP. brevicompactumand bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway ofP. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

scholarly journals Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

2012 ◽  
Vol 78 (14) ◽  
pp. 4908-4913 ◽  
Author(s):  
Bjarne Gram Hansen ◽  
Ewelina Mnich ◽  
Kristian Fog Nielsen ◽  
Jakob Blæsbjerg Nielsen ◽  
Morten Thrane Nielsen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Mycophenolic Acid ◽  
Polyketide Synthase ◽  
Fusion Gene ◽  
Bioinformatic Analysis ◽  
Terminal Region ◽  
Ring Closure ◽  
Content Type ◽  
Penicillium Brevicompactum

ABSTRACTMycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered inPenicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed inP. brevicompactumby obtaining full-length sequence of thempaDEcDNA prepared from the extracted RNA. Heterologous coexpression ofmpaCand the fusion genempaDEin the MPA-nonproducerAspergillus nidulansresulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressingmpaCand thempaDpart ofmpaDEshows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.

scholarly journals Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

2012 ◽  
Vol 78 (23) ◽  
pp. 8234-8244 ◽  
Author(s):  
Jennifer Gerke ◽  
Özgür Bayram ◽  
Kirstin Feussner ◽  
Manuel Landesfeind ◽  
Ekaterina Shelest ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Aspergillus Nidulans ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Cellular Protein ◽  
Protein Stabilization ◽  
Putative Gene ◽  
Content Type ◽  
Alternative Approach ◽  
Polyketide Synthase Gene

ABSTRACTThe genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungusAspergillus nidulansby deleting the conserved eukaryoticcsnE/CSN5deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). ThecsnE/CSN5gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.

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

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ming-Yueh Wu ◽  
Matthew E. Mead ◽  
Mi-Kyung Lee ◽  
George F. Neuhaus ◽  
Donovon A. Adpressa ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Secondary Metabolism ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Genetic Regulatory Networks ◽  
Asexual Development ◽  
Morphological Development ◽  
Vast Number ◽  
Content Type ◽  
Metabolic Remodeling

ABSTRACT 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 Aspergillus 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 Aspergillus nidulans. 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 A. nidulans 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 Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination. IMPORTANCE 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. Aspergillus 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 Aspergillus nidulans. 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 Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus.

scholarly journals Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

2015 ◽  
Vol 14 (9) ◽  
pp. 908-921 ◽  
Author(s):  
Nicole Bühler ◽  
Daisuke Hagiwara ◽  
Norio Takeshita
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Important Species ◽  
Content Type

ABSTRACT Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes ( oshA to oshE ) in the filamentous fungi Aspergillus nidulans . The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus , as well as A. nidulans . Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.

scholarly journals Identification and Regulation offusA, the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

2012 ◽  
Vol 78 (20) ◽  
pp. 7258-7266 ◽  
Author(s):  
Violeta Díaz-Sánchez ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Nitrogen Availability ◽  
Polyketide Synthase ◽  
High Sensitivity ◽  
Targeted Mutagenesis ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Wild Type ◽  
Sequence Comparisons ◽  
Content Type ◽  
Polyketide Synthase Gene

ABSTRACTFusarins are a class of mycotoxins of the polyketide family produced by differentFusariumspecies, including the gibberellin-producing fungusFusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in otherFusariumstrains, we have identified theF. fujikuroiorthologue, calledfusA. The participation offusAin fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by thefusAmRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of thefusAgene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, thefusAmutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions inFusarium oxysporum. ThefusAmutants, however, are not affected in their capacities to grow on plant tissues.

scholarly journals Transcriptomics-Aided Dissection of the Intracellular and Extracellular Roles of Microcystin in Microcystis aeruginosa PCC 7806

2014 ◽  
Vol 81 (2) ◽  
pp. 544-554 ◽  
Author(s):  
A. Katharina Makower ◽  
J. Merijn Schuurmans ◽  
Detlef Groth ◽  
Yvonne Zilliges ◽  
Hans C. P. Matthijs ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Wild Type Strain ◽  
Carbon Limitation ◽  
Light Conditions ◽  
Wild Type ◽  
Low Light ◽  
Content Type ◽  
Expression Of Genes ◽  
Polyketide Synthase Gene

ABSTRACTRecent studies have provided evidence for both intracellular and extracellular roles of the potent hepatotoxin microcystin (MC) in the bloom-forming cyanobacteriumMicrocystis. Here, we surveyed transcriptomes of the wild-type strainM. aeruginosaPCC 7806 and the microcystin-deficient ΔmcyBmutant under low light conditions with and without the addition of external MC of the LR variant (MC-LR). Transcriptomic data acquired by microarray and quantitative PCR revealed substantial differences in the relative expression of genes of the central intermediary metabolism, photosynthesis, and energy metabolism. In particular, the data provide evidence for a lower photosystem I (PSI)-to-photosystem II (PSII) ratio and a more pronounced carbon limitation in the microcystin-deficient mutant. Interestingly, only 6% of the transcriptional differences could be complemented by external microcystin-LR addition. This MC signaling effect was seen exclusively for genes of the secondary metabolism category. The orphan polyketide synthase gene cluster IPF38-51 was specifically downregulated in response to external MC-LR under low light. Our data suggest a hierarchical and light-dependent cross talk of secondary metabolites and support both an intracellular and an extracellular role of MC inMicrocystis.

scholarly journals The Aspergillus nidulansnucAEndoGHomologue Is Not Involved in Cell Death

2010 ◽  
Vol 10 (2) ◽  
pp. 276-283 ◽  
Author(s):  
Bárbara de Castro Pimentel Figueiredo ◽  
Patrícia Alves de Castro ◽  
Taísa Magnani Dinamarco ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Cell Death ◽  
Aspergillus Nidulans ◽  
Dna Fragmentation ◽  
Filamentous Fungi ◽  
Large Scale ◽  
Apoptosis Induction ◽  
Content Type ◽  
Endonuclease G ◽  
First Time

ABSTRACTUpon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if theAspergillus nidulanshomologue of the EndoG gene, namednucAEndoG, is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response inA. nidulans.

scholarly journals The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation

2012 ◽  
Vol 11 (3) ◽  
pp. 311-323 ◽  
Author(s):  
Yainitza Hernández-Rodríguez ◽  
Susan Hastings ◽  
Michelle Momany
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Early Growth ◽  
Content Type ◽  
Green Fluorescent ◽  
And Function ◽  
Cytoskeletal Elements ◽  
Germ Tubes

ABSTRACTIn yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungusAspergillus nidulansthere are five septin genes,aspA(an ortholog ofSaccharomyces cerevisiae CDC11),aspB(an ortholog ofS. cerevisiae CDC3),aspC(an ortholog ofS. cerevisiae CDC12),aspD(an ortholog ofS. cerevisiae CDC10), andaspE(found only in filamentous fungi). TheaspBgene was previously reported to be the most highly expressedAspergillus nidulansseptin and to be essential. Using improved gene targeting techniques, we found that deletion ofaspBis not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained inΔaspDand lost inΔaspAandΔaspCstrains. Interestingly, in theΔaspEmutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

scholarly journals Polyketide Synthase Gene Diversity within the Microbiome of the Sponge Arenosclera brasiliensis, Endemic to the Southern Atlantic Ocean

2012 ◽  
Vol 79 (5) ◽  
pp. 1598-1605 ◽  
Author(s):  
Amaro E. Trindade-Silva ◽  
Cintia P. J. Rua ◽  
Bruno G. N. Andrade ◽  
Ana Carolina Paulo Vicente ◽  
Genivaldo G. Z. Silva ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Polyketide Synthase ◽  
Gene Diversity ◽  
Marine Sponges ◽  
Type I ◽  
Content Type ◽  
Phylogenetic Reconstructions ◽  
Polyketide Synthase Gene ◽  
Exclusive Group ◽  
Southern Atlantic Ocean

ABSTRACTMicrobes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida spongeArenosclera brasiliensishosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome ofA. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel andA. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes inA. brasiliensisas well as the potential of its microbiome to serve as a source of new polyketides.

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