Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor

Rudolph, Martin; Vockenhuber, Michael-Paul; Suess, Beatrix

doi:10.1016/bs.mie.2014.10.036

Cited by 29 publications

(30 citation statements)

References 24 publications

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“…Based on a recent report that described the effect of upstream secondary structure within the 5′UTR on translation efficiency [27], we proposed that the aptamer domain of the theophylline riboswitch naturally forming a stem-loop upstream of the SD sequence after transcription augments the accessibility of the ribosome to the SD, eventually producing higher levels of GFP. These results are consistent with previously reported data in which the induced expression levels altered after combining different promoters with the same riboswitch [22, 23]. …”

Section: Discussionsupporting

confidence: 94%

“…Lower leaky expression level produced by riboswitch E was also observed in other theophylline-mediated inducible system in Streptomyces coelicolor , in which the leakiness of GUS level is 0.1 GU. However, the induced expression level of GUS in the Streptomyces system was lower than that in our construct [22]. …”

Section: Discussionmentioning

confidence: 99%

“…These unexpected results obviously indicate that the novel riboswitch-mediated element produced higher expression than previously reported, but at the cost of a decreased induction ratio. Riboswitches combined with different promoters display distinct properties in various bacteria [12, 18, 22, 23]. Therefore, it is worthwhile to assess the performance of riboswitch elements in the context of individual genetic backgrounds.…”

Section: Discussionmentioning

confidence: 99%

“…In several Cyanobacterial species, theophylline riboswitches combined with native strong promoter ConII led to high induction ratios mediated that were achieved at 2 or 3 mM inducer concentrations [13]. Even in other Gram-positive bacteria, such as Mycobacteria and Streptomyces , high levels of induced gene expression, controlled by riboswitch E* fused to other constitutive promoters, were obtained with treatment of no more than 5 mM theophylline [18, 22, 25]. …”

Section: Discussionmentioning

confidence: 99%

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Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

et al. 2016

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BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

et al. 2016

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“…Here, 10-40 mg of protein crude extract (cell disruption protocol, see section "SDS-PAGE and Western Blot Analysis") obtained from mycelium harvested from solid medium were used for the enzymatic reaction. The assay was performed as previously described (Rudolph et al, 2015).…”

Section: β-Glucuronidase Measurementmentioning

confidence: 99%

sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism

et al. 2020

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In contrast to transcriptional regulation, post-transcriptional regulation and the role of small non-coding RNAs (sRNAs) in streptomycetes are not well studied. Here, we focus on the highly conserved sRNA scr5239 in Streptomyces coelicolor. A proteomics approach revealed that the sRNA regulates several metabolic enzymes, among them phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the central carbon metabolism. The sRNA scr5239 represses pepck at the post-transcriptional level and thus modulates the intracellular level of phosphoenolpyruvate (PEP). The expression of scr5239 in turn is dependent on the global transcriptional regulator DasR, thus creating a feedback loop regulation of the central carbon metabolism. By post-transcriptional regulation of PEPCK and in all likelihood other targets, scr5239 adds an additional layer to the DasR regulatory network and provides a tool to control the metabolism dependent on the available carbon source.

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START: A Versatile Platform for Bacterial Ligand Sensing with Programmable Performances

Kim,

Seo,

Lim

et al. 2024

Advanced Science

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Recognition of signaling molecules for coordinated regulation of target genes is a fundamental process for biological systems. Cells often rely on transcription factors to accomplish these intricate tasks, yet the subtle conformational changes of protein structures, coupled with the complexity of intertwined protein interaction networks, pose challenges for repurposing these for bioengineering applications. This study introduces a novel platform for ligand‐responsive gene regulation, termed START (Synthetic Trans‐Acting Riboswitch with Triggering RNA). Inspired by the bacterial ligand sensing system, riboswitch, and the synthetic gene regulator, toehold switch, the START platform enables the implementation of synthetic biosensors for various ligands. Rational sequence design with targeted domain optimization yields high‐performance STARTs with a dynamic range up to 67.29‐fold and a tunable ligand sensitivity, providing a simple and intuitive strategy for sensor engineering. The START platform also exhibits modularity and composability to allow flexible genetic circuit construction, enabling seamless implementation of OR, AND, and NOT Boolean logic gates for multiple ligand inputs. The START design principle is capable of broadening the suite of synthetic biosensors for diverse chemical and protein ligands, providing a novel riboregulator chassis for synthetic biology and bioengineering applications.

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Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor

Cited by 29 publications

References 24 publications

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism

START: A Versatile Platform for Bacterial Ligand Sensing with Programmable Performances

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