scholarly journals Comparative genome and transcriptome analyses revealing interspecies variations in the expression of fungal biosynthetic gene clusters

2020 ◽  
Author(s):  
Hiroki Takahashi ◽  
Maiko Umemura ◽  
Masaaki Shimizu ◽  
Akihiro Ninomiya ◽  
Yoko Kusuya ◽  
...  
Keyword(s):  
Secondary Metabolism ◽  
Filamentous Fungi ◽  
Bioactive Compounds ◽  
Expression Patterns ◽  
Gene Clusters ◽  
Transcriptional Level ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fungal Secondary Metabolism ◽  
Conserved Gene

AbstractFilamentous fungi produce various bioactive compounds that are biosynthesized by a set of proteins encoded in biosynthetic gene clusters (BGCs). For an unknown reason, large parts of the BGCs are transcriptionally silent under laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional regulation of fungal secondary metabolism is not fully understood from an evolutionary viewpoint. To address this issue, we conducted comparative genomic and transcriptomic analyses using five closely related species of the Aspergillus section Fumigati: Aspergillus fumigatus, Aspergillus lentulus, Aspergillus udagawae, Aspergillus pseudoviridinutans, and Neosartorya fischeri. From their genomes, 298 secondary metabolite (SM) core genes were identified, with 27.4% to 41.5% being unique to a species. Compared with the species-specific genes, a set of section-conserved SM core genes was expressed at a higher rate and greater magnitude, suggesting that their expression tendency is correlated with the BGC distribution pattern. However, the section-conserved BGCs showed diverse expression patterns across the Fumigati species. Thus, not all common BGCs across species appear to be regulated in an identical manner. A consensus motif was sought in the promoter region of each gene in the 15 section-conserved BGCs among the Fumigati species. A conserved motif was detected in only two BGCs including the gli cluster. The comparative transcriptomic and in silico analyses provided insights into how the fungal SM gene cluster diversified at a transcriptional level, in addition to genomic rearrangements and cluster gains and losses. This information increases our understanding of the evolutionary processes associated with fungal secondary metabolism.Author summaryFilamentous fungi provide a wide variety of bioactive compounds that contribute to public health. The ability of filamentous fungi to produce bioactive compounds has been underestimated, and fungal resources can be developed into new drugs. However, most biosynthetic genes encoding bioactive compounds are not expressed under laboratory conditions, which hampers the use of fungi in drug discovery. The mechanisms underlying silent metabolite production are poorly understood. Here, we attempted to show the diversity in fungal transcriptional regulation from an evolutionary viewpoint. To meet this goal, the secondary metabolisms, at genomic and transcriptomic levels, of the most phylogenetically closely related species in Aspergillus section Fumigati were compared. The conserved biosynthetic gene clusters across five Aspergillus species were identified. The expression levels of the well-conserved gene clusters tended to be more active than the species-specific, which were not well-conserved, gene clusters. Despite highly conserved genetic properties across the species, the expression patterns of the well-conserved gene clusters were diverse. These findings suggest an evolutionary diversification at the transcriptional level, in addition to genomic rearrangements and gains and losses, of the biosynthetic gene clusters. This study provides a foundation for understanding fungal secondary metabolism and the potential to produce diverse fungal-based chemicals.

Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism

2021 ◽  
Author(s):  
Takayuki Motoyama ◽  
Tomoaki Ishii ◽  
Takashi Kamakura ◽  
Hiroyuki Osada
Keyword(s):  
Secondary Metabolism ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Tenuazonic Acid ◽  
Cellular Functions ◽  
Biosynthetic Gene Clusters ◽  
Chemical Library ◽  
Tea Production ◽  
Fungal Secondary Metabolism

Abstract The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Michalis Hadjithomas ◽  
I-Min Amy Chen ◽  
Ken Chu ◽  
Anna Ratner ◽  
Krishna Palaniappan ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Secondary Metabolism ◽  
Genomic Data ◽  
Gene Clusters ◽  
Metagenomic Data ◽  
Integrated Analysis ◽  
Analysis Tool ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Analysis Tools

ABSTRACTIn the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time inAlphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.IMPORTANCEIMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

scholarly journals Detecting and prioritizing biosynthetic gene clusters for bioactive compounds in bacteria and fungi

2019 ◽  
Vol 103 (8) ◽  
pp. 3277-3287 ◽  
Author(s):  
Phuong Nguyen Tran ◽  
Ming-Ren Yen ◽  
Chen-Yu Chiang ◽  
Hsiao-Ching Lin ◽  
Pao-Yang Chen
Keyword(s):  
Bioactive Compounds ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Bacteria And Fungi

Endophytic actinobacteria: Diversity, secondary metabolism and mechanisms to unsilence biosynthetic gene clusters

2017 ◽  
Vol 43 (5) ◽  
pp. 546-566 ◽  
Author(s):  
Raghavan Dinesh ◽  
Veeraraghavan Srinivasan ◽  
Sheeja T. E. ◽  
Muthuswamy Anandaraj ◽  
Hamza Srambikkal
Keyword(s):  
Secondary Metabolism ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Endophytic Actinobacteria

scholarly journals IMG-ABC v.5.0: an update to the IMG/Atlas of Biosynthetic Gene Clusters Knowledgebase

2019 ◽  
Author(s):  
Krishnaveni Palaniappan ◽  
I-Min A Chen ◽  
Ken Chu ◽  
Anna Ratner ◽  
Rekha Seshadri ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Secondary Metabolism ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Microbial Genomes ◽  
Initial Release ◽  
Systematic Identification ◽  
Biomedical Potential ◽  
Biosynthetic Machinery

Abstract Microbial secondary metabolism is a reservoir of bioactive compounds of immense biotechnological and biomedical potential. The biosynthetic machinery responsible for the production of these secondary metabolites (SMs) (also called natural products) is often encoded by collocated groups of genes called biosynthetic gene clusters (BGCs). High-throughput genome sequencing of both isolates and metagenomic samples combined with the development of specialized computational workflows is enabling systematic identification of BGCs and the discovery of novel SMs. In order to advance exploration of microbial secondary metabolism and its diversity, we developed the largest publicly available database of predicted BGCs combined with experimentally verified BGCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc-public). Here we describe the first major content update of the IMG-ABC knowledgebase, since its initial release in 2015, refreshing the BGC prediction pipeline with the latest version of antiSMASH (v5) as well as presenting the data in the context of underlying environmental metadata sourced from GOLD (https://gold.jgi.doe.gov/). This update has greatly improved the quality and expanded the types of predicted BGCs compared to the previous version.

scholarly journals Reclassification of the Specialized Metabolite Producer Pseudomonas mesoacidophila ATCC 31433 as a Member of the Burkholderia cepacia Complex

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
E. Joel Loveridge ◽  
Cerith Jones ◽  
Matthew J. Bull ◽  
Suzy C. Moody ◽  
Małgorzata W. Kahl ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Bioactive Compounds ◽  
Single Molecule ◽  
Complete Genome ◽  
Burkholderia Cepacia ◽  
Gene Clusters ◽  
Burkholderia Cepacia Complex ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type

ABSTRACT Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the β-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis. The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization. IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis. Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.

scholarly journals CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

2020 ◽  
Author(s):  
Indra Roux ◽  
Clara Woodcraft ◽  
Jinyu Hu ◽  
Rebecca Wolters ◽  
Cameron L.M. Gilchrist ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Gene Clusters ◽  
Laboratory Culture ◽  
Culture Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
Artificial Transcription Factors ◽  
Bioactive Secondary Metabolite

AbstractAccessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12aD156R-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.

scholarly journals CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi

2020 ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A.W. de Kok ◽  
Roel A.L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into an AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

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