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 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 Diversity of Secondary Metabolism in Aspergillus nidulans Clinical Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
M. T. Drott ◽  
R. W. Bastos ◽  
A. Rokas ◽  
L. N. A. Ries ◽  
T. Gabaldón ◽  
...  
Keyword(s):  
Secondary Metabolism ◽  
Aspergillus Nidulans ◽  
Reference Genome ◽  
Genetic Model ◽  
High Resolution Mass Spectrometry ◽  
Gene Clusters ◽  
Clinical Isolates ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Natural Diversity

ABSTRACT The filamentous fungus Aspergillus nidulans has been a primary workhorse used to understand fungal genetics. Much of this work has focused on elucidating the genetics of biosynthetic gene clusters (BGCs) and the secondary metabolites (SMs) they produce. SMs are both niche defining in fungi and of great economic importance to humans. Despite the focus on A. nidulans, very little is known about the natural diversity in secondary metabolism within this species. We determined the BGC content and looked for evolutionary patterns in BGCs from whole-genome sequences of two clinical isolates and the A4 reference genome of A. nidulans. Differences in BGC content were used to explain SM profiles determined using liquid chromatography–high-resolution mass spectrometry. We found that in addition to genetic variation of BGCs contained by all isolates, nine BGCs varied by presence/absence. We discovered the viridicatumtoxin BGC in A. nidulans and suggest that this BGC has undergone a horizontal gene transfer from the Aspergillus section Nigri lineage into Penicillium sometime after the sections Nigri and Nidulantes diverged. We identified the production of viridicatumtoxin and several other compounds previously not known to be produced by A. nidulans. One isolate showed a lack of sterigmatocystin production even though it contained an apparently intact sterigmatocystin BGC, raising questions about other genes and processes known to regulate this BGC. Altogether, our work uncovers a large degree of intraspecies diversity in BGC and SM production in this genetic model species and offers new avenues to understand the evolution and regulation of secondary metabolism. IMPORTANCE Much of what we know about the genetics underlying secondary metabolite (SM) production and the function of SMs in the model fungus Aspergillus nidulans comes from a single reference genome. A growing body of research indicates the importance of biosynthetic gene cluster (BGC) and SM diversity within a species. However, there is no information about the natural diversity of secondary metabolism in A. nidulans. We discovered six novel clusters that contribute to the considerable variation in both BGC content and SM production within A. nidulans. We characterize a diverse set of mutations and emphasize how findings of single nucleotide polymorphisms (SNPs), deletions, and differences in evolutionary history encompass much of the variation observed in nonmodel systems. Our results emphasize that A. nidulans may also be a strong model to use within-species diversity to elucidate regulatory cross talk, fungal ecology, and drug discovery systems.

scholarly journals The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production

2007 ◽  
Vol 6 (7) ◽  
pp. 1210-1218 ◽  
Author(s):  
Daren W. Brown ◽  
Robert A. E. Butchko ◽  
Mark Busman ◽  
Robert H. Proctor
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Fusarium Verticillioides ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Proteins ◽  
Wild Type ◽  
Polyketide Synthase Gene ◽  
Splice Forms ◽  
Self Protection

ABSTRACT Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Δfum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Δfum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

scholarly journals Functional Operons in Secondary Metabolic Gene Clusters in Glarea lozoyensis (Fungi, Ascomycota, Leotiomycetes)

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Qun Yue ◽  
Li Chen ◽  
Yan Li ◽  
Gerald F. Bills ◽  
Xinyu Zhang ◽  
...  
Keyword(s):  
Secondary Metabolism ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Transcriptional Level ◽  
Tetramic Acid ◽  
Protein Coding ◽  
Content Type ◽  
Eukaryotic Gene ◽  
Transcriptional Units ◽  
Gene Structures

ABSTRACT Operons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Protein-coding operons have not been reported in the Fungi even though they represent a very diverse kingdom of organisms. Here, we report a functional operon involved in the secondary metabolism of the fungus Glarea lozoyensis belonging to Leotiomycetes (Ascomycota). Two contiguous genes, glpks3 and glnrps7, encoding polyketide synthase and nonribosomal peptide synthetase, respectively, are cotranscribed into one dicistronic mRNA under the control of the same promoter, and the mRNA is then translated into two individual proteins, GLPKS3 and GLNRPS7. Heterologous expression in Aspergillus nidulans shows that the GLPKS3-GLNRPS7 enzyme complex catalyzes the biosynthesis of a novel pyrrolidinedione-containing compound, xenolozoyenone (compound 1), which indicates the operon is functional. Although it is structurally similar to prokaryotic operons, the glpks3-glnrps7 operon locus has a monophylogenic origin from fungi rather than having been horizontally transferred from prokaryotes. Moreover, two additional operons, glpks28-glnrps8 and glpks29-glnrps9, were verified at the transcriptional level in the same fungus. This is the first report of protein-coding operons in a member of the Fungi. IMPORTANCE Operons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Three operon-like gene structures for secondary metabolism that were discovered in the filamentous fungus Glarea lozoyensis are the first examples of protein-coding operons identified in a member of the Fungi. Among them, the glpks3-glnrps7 operon is responsible for the biosynthesis of xenolozoyenone, which is a novel tetramic acid-containing compound. Although structurally similar to prokaryotic operons, the glpks3-glnrps7 operon locus did not result from horizontal gene transfer from prokaryotes. In addition, operonlike structures have been predicted in silico to be common in other fungi. The common occurrence and operonlike structure in fungi provide evolutionary insight and essential data for eukaryotic gene transcription.

scholarly journals Genome-based survey of nonribosomal peptide synthetase and polyketide synthase gene clusters in type strains of the genus Microtetraspora

2016 ◽  
Vol 69 (9) ◽  
pp. 712-718 ◽  
Author(s):  
Hisayuki Komaki ◽  
Natsuko Ichikawa ◽  
Tomohiko Tamura ◽  
Akio Oguchi ◽  
Moriyuki Hamada ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Gene Clusters ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Polyketide Synthase Gene ◽  
Type Strains

scholarly journals Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42

2006 ◽  
Vol 188 (11) ◽  
pp. 4024-4036 ◽  
Author(s):  
Xiao-Hua Chen ◽  
Joachim Vater ◽  
Jörn Piel ◽  
Peter Franke ◽  
Romy Scholz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bacillus Amyloliquefaciens ◽  
Polyketide Synthase ◽  
Functional Characterization ◽  
Gene Clusters ◽  
Ionization Mass ◽  
Bioactive Natural Products ◽  
Flight Mass Spectrometry ◽  
Polyketide Synthase Gene ◽  
Bacterial Sources

ABSTRACT Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp 0), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.

scholarly journals Identification of polyketide synthase gene clusters in a phage P1-derived artificial chromosome library of a Philippine strain of Streptomyces sp. PCS3-D2

2019 ◽  
pp. 56-63
Author(s):  
Aileen Bayot Custodio ◽  
Edwin Plata Alcantara
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Genetic Manipulation ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Restriction Enzyme Digestion ◽  
Type I ◽  
Putative Gene ◽  
Streptomyces Sp ◽  
Type Iii

A phage P1-derived artificial chromosome (PAC) library was constructed from genomic DNA of Streptomyces sp. PCS3-D2. Polymerase chain reaction (PCR) screening of the PAC library revealed two clones, PAC16D and P222O, which were positively identified to harbor polyketide synthase (PKS) Type I and PKS Type III gene clusters, respectively. Restriction enzyme digestion showed that PAC16D and PAC222O contained a 130 kb and a 140 kb insert, respectively. Results of sequencing and bioinformatics analyses revealed that PAC16D comprised of a full-length PKS type I bafilomycin gene cluster while PAC222O harbored truncated siderophore and putative gene clusters as well as a complete PKS III biosynthetic gene cluster. The PKS III gene cluster had three genes similar to alkyl resorcinol biosynthetic genes, however majority of the novel gene cluster had little similarity to known PKS Type III gene clusters. The successful cloning and identification of these gene clusters from Streptomyces sp. PCS3-D2 serve as the jump off point to further genetic manipulation in order to produce the insecticidal natural product in a heterologous host.

scholarly journals Draft Genome Assembly of the Entomopathogenic Bacterium Photorhabdus luminescens subsp. sonorensis Caborca

2019 ◽  
Vol 8 (36) ◽  
Author(s):  
Duy An Duong ◽  
Patricia Espinosa-Artiles ◽  
Rousel A. Orozco ◽  
István Molnár ◽  
S. Patricia Stock
Keyword(s):  
Secondary Metabolism ◽  
Genome Assembly ◽  
Draft Genome ◽  
Gene Clusters ◽  
Insect Species ◽  
Photorhabdus Luminescens ◽  
Putative Gene ◽  
Content Type ◽  
Draft Genome Assembly ◽  
Wide Range

Photorhabdus luminescens subsp. sonorensis strain Caborca is an entomopathogenic bacterium with a dual lifestyle, namely, as a mutualist of the Heterorhabditis sonorensis nematode and a pathogen to a wide range of insect species. The genome assembly, in 231 contigs, is 5.2 Mbp long and includes 25 putative gene clusters for secondary metabolism.

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