Biosynthetic Gene Cluster regulatory sequence library profiled in S. albidoflavus
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 550
ORGANISM(S): Streptomyces albidoflavus
SUBMITTER: Jimin Park
PROVIDER: E-MTAB-9223 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.
2019-05-01 | E-MTAB-7715 | biostudies-arrayexpress
Project description:Characterization of a metagenomic regulatory sequence library derived from M. xanthus, E. coli, and O. urethralis genomes in strains expressing different RpoD ortholog variants. Targeted DNA and RNA seq used to profile relative DNA and RNA abundances, respectively of each regulatory sequence construct in the library.
2022-06-07 | E-MTAB-9111 | biostudies-arrayexpress
Project description:An improved understanding of the genome-wide regulation of natural compound biosynthesis in bacterial producers may accelerate the discovery of novel biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed the dynamic temporal variation of transcriptional activity, indicating that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and nonribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these surges in BGC transcriptional activity were associated with boosts in the production of known natural compounds, indicating that their biosynthesis was crucially regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provided unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under meager conditions for bacterial growth. The close association between BGC transcription and compound production suggested that the molecular manipulation of transcriptional activity may be a viable strategy to increase compound yields from myxobacterial producer strains, warranting increased efforts to develop genetic engineering tools for these organisms.
2023-05-03 | GSE217497 | GEO
Project description:Background: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterization work we found that this strain is able to produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and shows anti-tumor activity in RAS-dependent cancer cells. Results: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is s indeed responsible for rasfonin biosynthesis. Conclusions: The results of this study lay the ground for molecular biology focused research in Cephalotrichum gorgonifer. Furthermore, strain engineering and heterologous expression of the rasfonin BGC is now possible which facilitates the construction of high producing strains or the synthesis of rasfonin derivates for diverse applications.
2022-11-11 | GSE217303 | GEO
Project description:Employing multi-omics methods for overexpression of griseusins in Streptomyces sp. CA-25628. This set of data proves that the introduction of the SARPs regulator activates the production of griseusins ( [M+H]+= 749.2589, 586.2288, 796.2771), the inactivation of the griseusin expression using CRISPR-cBEST base editing, and the heterologous expression of the BGC responsible for their production, complemented with the SARPs regulator, activates their production in S. albus J1074.
2021-05-21 | MSV000087475 | MassIVE
Project description:The functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to test the impact of individual microRNAs on the growth of FLT3-ITD positive leukemia cells. This approach identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote tumor cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. Our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease. Loss-of-function CRISPR-Cas9 screen identifies genes whose loss leads to increased or decreased FLT3-ITD+ cell growth over 23 day time-course
2016-07-01 | E-GEOD-71539 | biostudies-arrayexpress
Project description:BGC-823 Cells: Control vs. BGC-823 Cells METTL3 Overexpressed
| PRJNA881250 | ENA
Project description:Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here, we present a novel approach utilising a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and NADPH-dependent terminal thioreductases (TRs), previously untapped in engineered PKSs. Reduction of the terminal aldehyde with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specific transaminases enables production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A (CoA)–specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl-CoA–specific ATs enables production of branched-chain diols and amino alcohols. In total, we demonstrated production of nine 1,3-diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl-1,3-hexanediol), six amino alcohols, and two carboxylic acids using our PKS platform in Streptomyces albus. Finally, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g/L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform.
2024-12-02 | PXD055149 | Pride