Abstract
Summary
Genes involved in biological pathways are often collocalised in gene clusters, the comparison of which can give valuable insights into their function and evolutionary history. ...However, comparison and visualization of gene cluster similarity is a tedious process, particularly when many clusters are being compared. Here, we present clinker, a Python based tool and clustermap.js, a companion JavaScript visualization library, which used together can automatically generate accurate, interactive, publication-quality gene cluster comparison figures directly from sequence files.
Availability and implementation
Source code and documentation for clinker and clustermap.js is available on GitHub (github.com/gamcil/clinker and github.com/gamcil/clustermap.js, respectively) under the MIT license. clinker can be installed directly from the Python Package Index via pip.
Supplementary information
Supplementary data are available at Bioinformatics online.
As structure prediction methods are generating millions of publicly available protein structures, searching these databases is becoming a bottleneck. Foldseek aligns the structure of a query protein ...against a database by describing tertiary amino acid interactions within proteins as sequences over a structural alphabet. Foldseek decreases computation times by four to five orders of magnitude with 86%, 88% and 133% of the sensitivities of Dali, TM-align and CE, respectively.
The burnettramic acids are a new class of antibiotics from an Australian fungus Aspergillus burnettii. The rare bolaamphiphilic scaffold consists of β-d-mannose linked to a pyrrolizidinedione unit ...via a 26-carbon chain. The most abundant metabolite displayed potent in vitro antifungal activity. Comparative genomics identified the hybrid PKS-NRPS bua gene cluster, which was verified by heterologous pathway reconstitution in Aspergillus nidulans.
1-Benzazepine is a pharmaceutically important scaffold but is rare among natural products. Nanangelenin A (1), containing an unprecedented ...3,4-dihydro-1-benzazepine-2,5-dione-N-prenyl-N-acetoxy-anthranilamide scaffold, was isolated from a novel species of Australian fungus, Aspergillus nanangensis. Genomic and retrobiosynthetic analyses identified a putative nonribosomal peptide synthetase (NRPS) gene cluster (nan). The detailed biosynthetic pathway to 1 was established by heterologous pathway reconstitution in A. nidulans, which led to biosynthesis of intermediates nanagelenin B–F (2–5 and 7). We demonstrated that the NRPS NanA incorporates anthranilic acid (Ant) and l-kynurenine (l-Kyn), which is supplied by a dedicated indoleamine-2,3-dioxygenase NanC encoded in the gene cluster. Using heterologous in vivo assays and mutagenesis, we demonstrated that the C-terminal condensation (CT) and thiolation (T3) domains of NanA are responsible for the regioselective cyclization of the tethered Ant-l-Kyn dipeptide to form the unusual benzazepine scaffold in 1. We also showed that NanA-CT catalyzes the regioselective cyclization of a surrogate synthetic substrate, Ant-l-Kyn-N-acetylcysteamine, to give the benzazepine scaffold, while spontaneous cyclization of the dipeptide yielded the alternative kinetically favored benzodiazepine scaffold. The discovery of 1 and the characterization of NanA have expanded the chemical and functional diversities of fungal NRPSs.
In phylogenomics the evolutionary relationship of organisms is studied by their genomic information. A common approach to phylogenomics is to extract related genes from each organism, build a ...multiple sequence alignment and then reconstruct evolution relations through a phylogenetic tree. Often a set of highly conserved genes occurring in single-copy, called core genes, are used for this analysis, as they allow efficient automation within a taxonomic clade. Here we introduce the Universal Fungal Core Genes (UFCG) database and pipeline for genome-wide phylogenetic analysis of fungi. The UFCG database consists of 61 curated fungal marker genes, including a novel set of 41 computationally derived core genes and 20 canonical genes derived from literature, as well as marker gene sequences extracted from publicly available fungal genomes. Furthermore, we provide an easy-to-use, fully automated and open-source pipeline for marker gene extraction, training and phylogenetic tree reconstruction. The UFCG pipeline can identify marker genes from genomic, proteomic and transcriptomic data, while producing phylogenies consistent with those previously reported, and is publicly available together with the UFCG database at https://ufcg.steineggerlab.com.
Accessing 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 BGCs 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, multigene 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.
Fungi are a rich source of bioactive small molecules. However, the large number of biosynthetic gene clusters (BGCs) encoding these molecules in their genomes suggests their biosynthetic potential is ...far greater than we previously appreciated. The mining of fungal genomes therefore holds great promise for the discovery of new chemical entities for pharmaceutical and agricultural applications. As more and more fungal genomes become available, the accompanying number of BGCs is quickly becoming unmanageable. Along with improving molecular genetic tools to accelerate the translation of BGCs to small molecules, we must devise strategies to prioritise BGCs most likely to encode the biosynthesis of novel small molecules and molecules with new or improved bioactivities or functions. In this perspective, we discuss existing and emerging strategies for prioritisation of BGCs to increase the odds of fruitful genome mining in fungi.
A perspective on existing and emerging strategies for the prioritisation of secondary metabolite biosynthetic gene clusters (BGCs) to increase the odds of fruitful mining of fungal genomes.
Genes involved in coordinated biological pathways, including metabolism, drug resistance and virulence, are often collocalized as gene clusters. Identifying homologous gene clusters aids in the study ...of their function and evolution, however, existing tools are limited to searching local sequence databases. Tools for remotely searching public databases are necessary to keep pace with the rapid growth of online genomic data.
Here, we present cblaster, a Python-based tool to rapidly detect collocated genes in local and remote databases. cblaster is easy to use, offering both a command line and a user-friendly graphical user interface. It generates outputs that enable intuitive visualizations of large datasets and can be readily incorporated into larger bioinformatic pipelines. cblaster is a significant update to the comparative genomics toolbox.
cblaster source code and documentation is freely available from GitHub under the MIT license (github.com/gamcil/cblaster).
Supplementary data are available at
online.
The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. ...Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1–3) with variable numbers of “GLKLAF” core peptide repeats corresponding to the victorin peptide backbone. vicA1–3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.
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