50 research outputs found
Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)8 isomerase evolutionary intermediates from a diverse metagenome
MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters
Microbial Biotechnolog
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Minimum Information about a Biosynthetic Gene cluster
A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog
Expression, purification and preliminary crystallographic analysis of phosphoribosyl isomerase (PriA)from streptomyces coelicolor.
The priA gene encoding the enzyme phosphoribosyl isomerase from Streptomyces coelicolor, a novel bifunctional enzyme involved in both histidine and tryptophan biosynthesis, was heterologously expressed and purified in Escherichia coli as an N-terminal His-tag fusion. The purified recombinant enzyme was crystallized using the hanging-drop method in 1.50 M ammonium sulfate and 100 mM sodium citrate pH 4.8. Crystals were obtained of up to 0.05x0.05x0.3 mm in size. A full data set to 2 Angstrom resolution was collected at the ESRF beamline ID14-1 and space group P3(1,2)21 was assigned, with unit-cell parameters a=65.1, c=104.7 Angstrom
Distinct extracytoplasmic siderophore binding proteins recognize ferrioxamines and ferricoelichelin in streptomyces coelicolor A3(2)
Under iron limitation, the Gram-positive bacterium Streptomyces coelicolor A3(2) excretes three siderophores of the hydroxamate type: desferrioxamine B, desferrioxamine E, and coelichelin. These sequester iron from insoluble ferric hydroxides, and the resulting ferric complexes are believed to be transported into the cell via siderophore-binding proteins (SBPs) associated with ATP-binding cassette (ABC) transporters. Previous studies indicated that some of the genes in the desferrioxamine (des) and coelichelin (cch) biosynthetic clusters encode ABC transporter components required for efficient uptake of ferrioxamine E and ferricoelichelin, respectively, and a third ABC transporter gene cluster (cdt), not associated with siderophore biosynthesis genes, was implicated in the import of ferrioxamine B. In this study, the putative SBPs associated with these three gene clusters, DesE, CchF, and CdtB, were recombinantly overproduced in Escherichia coli and purified to homogeneity, and their binding affinity for cognate siderophores and noncognate siderophores was examined using fluorescence and circular dichroism spectroscopy. DesE was found to bind all of the ferric-tris-hydroxamates tested except ferricoelichelin, while CchF was found to bind only ferricoelichelin efficiently, providing further evidence that the cch cluster is a complete siderophore biosynthesis export uptake gene cluster. The picture was more complicated for CdtB, because it was found to be unstable in solution but was found to bind both ferrioxamine B and ferricoelichelin with high affinity. This was surprising because the cch cluster was previously reported to be necessary for efficient ferricoelichelin uptake. The remarkable specificity of the DesE and CchF proteins for different ferric-tris-hydroxamates raises intriguing questions about the molecular basis of their substrate specificity
Liver transplantation due to Fulminant Liver Failure secondary to hepatic submassive necrosis in a patient treated with Orlistat
A computational framework for systematic exploration of biosynthetic diversity from large-scale genomic data
Genome mining has become a key technology to explore and exploit natural product diversity through the identification and analysis of biosynthetic gene clusters (BGCs). Initially, this was performed on a single-genome basis; currently, the process is being scaled up to large-scale mining of pan-genomes of entire genera, complete strain collections and metagenomic datasets from which thousands of bacterial genomes can be extracted at once. However, no bioinformatic framework is currently available for the effective analysis of datasets of this size and complexity. Here, we provide a streamlined computational workflow, tightly integrated with antiSMASH and MIBiG, that consists of two new software tools, BiG-SCAPE and CORASON. BiG-SCAPE facilitates rapid calculation and interactive visual exploration of BGC sequence similarity networks, grouping gene clusters at multiple hierarchical levels, and includes a 'glocal' alignment mode that accurately groups both complete and fragmented BGCs. CORASON employs a phylogenomic approach to elucidate the detailed evolutionary relationships between gene clusters by computing high-resolution multi-locus phylogenies of all BGCs within and across gene cluster families (GCFs), and allows researchers to comprehensively identify all genomic contexts in which particular biosynthetic gene cassettes are found. We validate BiG-SCAPE by correlating its GCF output to metabolomic data across 403 actinobacterial strains. Furthermore, we demonstrate the discovery potential of the platform by using CORASON to comprehensively map the phylogenetic diversity of the large detoxin/rimosamide gene cluster clan, prioritizing three new detoxin families for subsequent characterization of six new analogs using isotopic labeling and analysis of tandem mass spectrometric data
A computational framework to explore large-scale biosynthetic diversity
Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the 'biosynthetic gene similarity clustering and prospecting engine' (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the 'core analysis of syntenic orthologues to prioritize natural product gene clusters' (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues