Combinatorial effect of ARTP mutagenesis and ribosome engineering on an industrial Streptomyces albus strain S12 for enhanced biosynthesis of salinomycin
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ABSTRACT:
PROVIDER: PRJNA551961 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9622380.fastq.gz | Fastqsanger.gz | |||
| SRR9622381.fastq.gz | Fastqsanger.gz | |||
| SRR9622382.fastq.gz | Fastqsanger.gz |
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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:Salinomycin, an important polyketide, has been widely utilized in agriculture to inhibit growth of pathogenic bacteria. In addition, salinomycin has great potential in treatment of cancer cells. Due to inherited characteristics and beneficial potential, its demand is also inclining. Therefore, there is an urgent need to increase the current high demand of salinomycin. In order to obtain a high-yield mutant strain of salinomycin, the present work has developed an efficient breeding process of Streptomyces albus by using atmospheric and room temperature plasma (ARTP) combined with ribosome engineering. In this study, we investigate the presented method as it has the advantage of significantly shortening mutant screening duration by using an agar block diffusion method, as compared to other traditional strain breeding methods. As a result, the obtained mutant Tet30Chl25 with tetracycline and chloramphenicol resistance provided a salinomycin yield of 34,712 mg/L in shake flask culture, which was over 2.0-fold the parental strain S12. In addition, comparative transcriptome analysis of low and high yield mutants, and a parental strain revealed the mechanistic insight of biosynthesis pathways, in which metabolic pathways including butanoate metabolism, starch and sucrose metabolism and glyoxylate metabolism were closely associated with salinomycin biosynthesis. Moreover, we also confirmed that enhanced flux of glyoxylate metabolism via overexpression gene of isocitrate lyase (icl) promoted salinomycin biosynthesis. Based on these results, it has been successfully verified that the overexpression of crotonyl-CoA reductase gene (crr) and transcriptional regulator genes (orf 3 and orf 15), located in salinomycin synthesis gene cluster, is possibly responsible for the increase in salinomycin production in a typical strain Streptomyces albus DSM41398. Conclusively, a tentative regulatory model of ribosome engineering combined with ARTP in S. ablus is proposed to explore the roles of transcriptional regulators and stringent responses in the biosynthesis regulation of salinomycin.
| S-EPMC6733881 | biostudies-literature
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:Pseudomonas putida S12 is an inherently solvent-tolerant strain and constitutes a promising platform for biotechnology applications in whole-cell biocatalysis of aromatic compounds. The genome of P. putida S12 consists of a 5.8 Mbp chromosome and a 580 kbp megaplasmid pTTS12. pTTS12 encodes several genes which enable the tolerance to various stress conditions, including the main solvent efflux pump SrpABC. Removal (curing) of megaplasmid pTTS12 and subsequent loss of solvent efflux pump SrpABC caused a significant reduction in solvent tolerance of the resulting strain. In this study, we succeeded in restoring solvent tolerance in the megaplasmid-cured P. putida S12 using adaptive laboratory evolution (ALE) and molecular analysis to investigate the intrinsic solvent tolerance of P. putida S12. RNA-seq was performed to study the global transcriptomic response of the solvent-adapted plasmid-cured P. putida S12 in the presence of toluene. This analysis revealed the downregulation of ATP synthase, flagella and other RND efflux pumps, which indicates the importance of maintaining proton motive force during solvent stress.
2021-02-24 | GSE144045 | GEO
Project description:Here, we established a successive Fe0-enhanced microbe system to remove azo dye (a typical organic pollutant) by Shewanella decolorationis S12 (S. decolorationis S12, an effective azo dye degradation bacterium) and examined the gene expression time course (10, 30, 60, and 120 min) in whole genome transcriptional level. Comparing with the treatment without ZVI, approximately 8% genes affiliated with 10 different gene expression profiles in S. decolorationis S12 were significantly changed in 120 min during the ZVI-enhanced microbial azo reduction. Intriguingly, MarR transcriptional factor might play a vital role in regulating ZVI-enhanced azo reduction in the aspect of energy production, iron homeostasis, and detoxification. Further investigation showed that induced [Ni-Fe] H2ase genes (hyaABCDEF) and azoreductase genes (mtrABC-omcA) contributed to ZVI-enhanced energy production, while reduced iron uptake (hmuVCB and feoAB), induced sulfate assimilation (cysPTWA) and cysteine biosynthesis (cysM) related genes were essential to iron homeostasis and detoxification. This study disentangles underlying mechanisms of ZVI-enhanced azo reduction in S. decolorationis S12 and lays a foundation for further optimization of integrated ZVI-microbial system for efficient organic pollution treatment.
2021-10-01 | GSE160493 | GEO
Project description:Pseudomonas putida S12 Genome sequencing
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Project description:Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450BM3, pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.
| S-EPMC5435451 | biostudies-literature
Project description:Shewanella decolorationis S12 Genome sequencing and assembly
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