Metabolic model for diversity-generating biosynthesis.
Ontology highlight
ABSTRACT:
SUBMITTER: Tianero MD
PROVIDER: S-EPMC4763782 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We present the RAVEN (Reconstruction, Analysis and Visualization of Metabolic Networks) Toolbox: a software suite that allows for semi-automated reconstruction of genome-scale models. It makes use of published models and/or the KEGG database, coupled with extensive gap-filling and quality control features. The software suite also contains methods for visualizing simulation results and omics data, as well as a range of methods for performing simulations and analyzing the results. The software is a useful tool for system-wide data analysis in a metabolic context and for streamlined reconstruction of metabolic networks based on protein homology. The RAVEN Toolbox workflow was applied in order to reconstruct a genome-scale metabolic model for the important microbial cell factory Penicillium chrysogenum Wisconsin54-1255. The model was validated in a bibliomic study of in total 440 references, and it comprises 1471 unique biochemical reactions and 1006 ORFs. It was then used to study the roles of ATP and NADPH in the biosynthesis of penicillin, and to identify potential metabolic engineering targets for maximization of penicillin production.
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Project description:In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.
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Project description:Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by accelerating the evolution of target genes through a specialized, error-prone, reverse transcription process. First identified in a Bordetella phage (BPP-1), which mediates the phage tropism specificity by generating variability in an involved gene, DGRs were predicted to be present in a larger collection of viral and bacterial species. A minimal DGR system is comprised of a reverse transcriptase (RTase) gene, a template sequence (TR) and a variable region (VR) within a target gene. We developed a computational tool, DGRscan, to allow either de novo identification (based on the prediction of potential template-variable region pairs) or similarity-based searches of DGR systems using known template sequences as the reference. The application of DGRscan to the human microbiome project (HMP) datasets resulted in the identification of 271 non-redundant DGR systems, doubling the size of the collection of known DGR systems. We further identified a large number of putative target genes (651, which share no more than 90% sequence identity at the amino acid level) that are potentially under diversification by the DGR systems. Our study provides the first survey of the DGR systems in the human microbiome, showing that the DGR systems are frequently found in human-associated bacterial communities, although they are of low incidence in individual genomes. Our study also provides functional clues for a large number of genes (reverse transcriptases and target genes) that were previously annotated as proteins of unknown functions or nonspecific functions.
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Project description:Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.
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Project description:Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.
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Project description:Mammalian development is an intricate process regulated by multiple gene isoforms and their epigenetic states, which are yet undefined. Using integrative massive parallel sequencing and bioinformatics approach, we built genome-wide inventory of transcript variants, their promoters and histone modification states during normal development, using mouse cerebellum as model system. The data we integrated consists of 29,589 (4,792 novel) promoters that transcribe 61,525 (12,796 novel) distinct mRNAs, corresponding to 14,508 protein-coding and 9,862 non-coding genes. While 68% of the multi-transcript genes exhibit alternative splicing, 78% use alternative transcriptional events that are regulated during cerebellar development through H3K4me3 and H3K27me3. The data presented highlight the magnitude of alternative promoters and transcriptional termination as major source of transcriptome diversity along with alternative splicing. We also show that alternative promoters differentially activated during normal cerebellar development are aberrantly used in medulloblastoma, emphasizing the importance of studying gene regulation and function at the isoform-level. Study of transcriptome diversity in cerebellar development. We performed mRNA-seq and ChIP-seq experiments. mRNA-seq were performed on total RNA isolated from two P0, P5, P15 and adult cerebellum using 10 microgram of total RNA as the starting material for sequencing library prep. For each stage, mRNA-seq data was obtained from two lanes of a flowcell. For ChIP-seq experiments, solubilized chromatin was prepared by pooling multiple cerebellum tissues (3-18) and then ChIP-enriched DNA (using anti-RNAP II, anti-H3K4me3, anti-H3K27me3, or negative control IgG antibodies) were isolated. 10 microgram of ChIP-enriched DNA was used to prepare the ChIP-seq library for sequencing in a single lane.
2011-06-07 | E-GEOD-23525 | biostudies-arrayexpress