Systems metabolic engineering of Streptomyces albus for advanced production of the reverse antibiotic nybomycin
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ABSTRACT:
ORGANISM(S): Streptomyces albidoflavus
PROVIDER: GSE240471 | GEO | 2023/08/10
REPOSITORIES: GEO
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Project description:Systems metabolic engineering of Streptomyces albus for advanced production of the reverse antibiotic nybomycin
| PRJNA1003853 | ENA
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:To evaluate the DNA binding of FscRI in Streptomyces albus S4 in vivo ChIP-Seq experiments were carried out on dusing anti-FLAG antibodies against 3xFLAG-FscRI protein using two biological replicates. The wild type strain was used as a control in experiments using the anti-FLAG antibodies as well as a DNA only control.
2016-11-14 | E-MTAB-5122 | biostudies-arrayexpress
Project description:Streptomyces sp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline production in these microbes. Here, we studied the production of pamamycins, macrodiolide homologues with a high activity against multi-resistant pathogenic microbes, using recombinant S. albus J1074/R2. Talc particles of micrometer size added to submerged cultures of the recombinant strain tripled pamamycin production up to 50 mg L-1. Furthermore, they strongly affected morphology, reduced the size of cell pellets, formed by the filamentous microbe during the process, up to six-fold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and metabolome of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3341 genes (56%), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to log2 10-fold). Furthermore, the microparticles perturbed genes encoding for central catabolism and CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased four-fold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. Our findings are straightforward to further develop pamamycins into antituberculosis leads. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes.
2020-07-25 | GSE155008 | GEO
Project description:To evaluate the DNA binding of AntA in Streptomyces albus S4, in vivo ChIP-Seq experiments were performed 3xFLAG-AntA and anti-FLAG antibodies using two biological replicates. The wild type strain was used as a control in experiments using the anti-FLAG antibodies as well as a DNA only control (which were published previously, E-MTAB-5122).
2019-02-28 | E-MTAB-7700 | biostudies-arrayexpress
Project description:Streptomyces albus S12, TK and Tet30Chl25 are the parental strain , low-yield and high-yield of salinomycin mutant obtained by ARTP and ribosome engineering ,respectively. There are total 1602 differentially expressed genes (DEGs) show differences in expression between the mutant strain TK, Tet30Chl25 and the initial strain S12. KEGG pathway analysis of differentially expressed genes (DEGs) between the mutant strain TK, Tet30Chl25 and the initial strain S12 show that the relevant differential pathways affecting salinomycin production were mainly related to butanoate metabolism, starch and sucrose metabolism, glyoxylate metabolism. Besides , the transcription of genes in the salinomycin biosynthesis gene cluster and the transcription level of related genes in the precursors biosynthesis pathway were more active in the high-yield salinomycin production strain Tet30Chl25. Furthermore, the transcription level ribosomal protein, string response, two component system and sigma factors are more active in high-yield of salinomycin mutants and that may involve in regulation of salinomycin biosynthesis and may account for the high-yield of salinomycin.
2019-10-01 | GSE133580 | GEO
Project description:Background: Pamamycins macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are still low and pamamycins are typically formed as crude mixture which mainly contains smaller derivatives. Therefore, strategies that enable a more efficient production of pamamycins with increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis to enhance pamamycin production in the heterologous host S. albus J1074/R2. Results: Supplemented with L-valine, the recombinant pamamycin producer revealed a threefold increased pamamycin titer of about 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA esters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, the presence of L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was activated, restricting production to the post L-valine phase. A null mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis and accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the regulator mutant enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663, almost 20-fold more than the wild type. Conclusions: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.
2021-04-01 | GSE168592 | GEO
Project description:Streptomyces albus Genome sequencing and assembly
| PRJNA561672 | ENA