Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Sung, Minhui; Yoo, Seung Min; Ren, Jun; Lee, Jae Eun; Lee, Sang Yup; Na, Dokyun

doi:10.1007/s12257-016-0245-y

Cited by 10 publications

(7 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, the response obtained from the plasmids containing Streptomyces -derived promoters ( scbRp , scbAp and cpkOp ) results in relatively low maximum expression of GFP, as opposed to the high levels of expression obtained from luxp , with an approximately 2-fold difference of relative strength between them (Figure ). In synthetic microorganisms, especially in those designed to produce specialty chemicals, fine-tuning and balancing of all proteins in the biosynthetic pathway has been previously shown to play a crucial role in the titers produced, , and overly strong induction of the proteins involved in the biosynthetic pathway can result in an impairment of the intracellular metabolite fluxes. The combination of the Streptomyces- derived promoters with the luxp can provide the means to balance and regulate refactored pathways.…”

Section: Results and Discussionmentioning

confidence: 99%

“…8). In 288 synthetic microorganisms, especially in those designed to produce speciality chemicals, fine tuning and balancing of all proteins in the biosynthetic pathway has been previously shown to play a crucial role in the titres produced 58,59 , and overly strong induction of the proteins involved in the biosynthetic pathway can result in an impairment of the intracellular metabolite fluxes. The combination of the Streptomyces-derived promoters with the luxp can provide the means to balance and regulate refactored pathways.…”

Section: E) Crosstalk Evaluation Between the Gbl And The Ahl Systemsmentioning

confidence: 99%

See 1 more Smart Citation

Orthogonal Regulatory Circuits for Escherichia coli Based on the γ-Butyrolactone System of Streptomyces coelicolor

et al. 2018

View full text Add to dashboard Cite

Chemically inducible transcription factors are widely used to control gene expression of synthetic devices. The bacterial quorum sensing system is a popular tool to achieve such control. However, different quorum sensing systems have been found to cross-talk, both between themselves and with the hosts of these devices, and they are leaky by nature. Here we evaluate the potential use of the γ-butyrolactone system from Streptomyces coelicolor A3(2) M145 as a complementary regulatory circuit. First, two additional genes responsible for the biosynthesis of γ-butyrolactones were identified in S. coelicolor M145 and then expressed in E. coli BL21 under various experimental conditions. Second, the γ-butyrolactone receptor ScbR was optimized for expression in E. coli BL21. Finally, signal and promoter crosstalk between the γ-butyrolactone system from S. coelicolor and quorum sensing systems from Vibrio fischeri and Pseudomonas aeruginosa was evaluated. The results show that the γ-butyrolactone system does not crosstalk with the quorum sensing systems and can be used to generate orthogonal synthetic circuits.

show abstract

Section: Results and Discussionmentioning

confidence: 99%

Section: E) Crosstalk Evaluation Between the Gbl And The Ahl Systemsmentioning

confidence: 99%

Orthogonal Regulatory Circuits for Escherichia coli Based on the γ-Butyrolactone System of Streptomyces coelicolor

et al. 2018

View full text Add to dashboard Cite

show abstract

“…One strategy is to modulate the target mRNA-binding ability of synthetic sRNA, which can be achieved by changing the sequence of the target-binding region within synthetic sRNA (Na et al, 2013). The other strategy is to vary the synthetic sRNA expression level (Sung et al, 2016) by employing promoters of different strengths and/or plasmids of different copy numbers. In the first strategy, synthetic sRNAs with different binding strengths can be designed based on calculation of the binding affinities of synthetic sRNAs using several programs, such as UNAfold (Markham and Zuker, 2008) and ViennaRNA (Lorenz et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Gene Expression Knockdown by Modulating Synthetic Small RNA Expression in Escherichia coli

et al. 2017

Self Cite

View full text Add to dashboard Cite

Escherichia coli gene expression knockdown using synthetic small RNA (sRNA) can be fine-tuned by altering sRNA sequences to modulate target mRNA-binding ability, but this requires thorough checking for off-target effects. Here, we present an sRNA gene expression knockdown system fine-tuned by using different promoters to modulate synthetic sRNA abundance. Our approach entails selecting knockdown target genes resulting from in silico flux response analysis and those related to product biosynthesis then screening strains transformed with a library of synthetic sRNA-promoter combinations for enhanced production. We engineered two E. coli strains, both utilizing fine-tuned repression of argF and glnA through our approach; one produced putrescine (42.3 ± 1.0 g/L) and the other L-proline (33.8 ± 1.6 g/L) by fed-batch culture. Fine-tuned gene knockdown by controlling sRNA abundance will be useful for rapid design of microbial strains through simultaneously optimizing expression of multiple genes at a systems level, as it overcomes the difficulties of constructing and testing many different sRNAs and checking their cross-reactivity.

show abstract

“…strengths (Noh et al, 2019;Sung et al, 2016;Na et al, 2013). The construction of robust synthetic gene circuits, coupled with reliable sRNA-based approaches for their regulation, provides a framework for development of novel applications in the personalized medicine, RNA-based diagnostics and therapeutics, and improve strains for expression (Pardee et al, 2016;Harimoto et al, 2022;Noh et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Tailored synthetic sRNAs dynamically tune multilayer genetic circuits

Sanchez¹,

Klopprogge²,

Zimmermann³

et al. 2022

Preprint

View full text Add to dashboard Cite

Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose a rational design of de novo small RNAs (sRNAs) to dynamically modulate gene expression within a broad range, from high, medium to low repression, and implemented them in Escherichia coli. We designed multiple multilayer genetic circuits, in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein. Our approach harnesses the intrinsic RNA-interference pathway in E. coli to exert dynamic and modular control of the multilayer genetic circuits. The sRNAs were designed to target TFs instead of the reporter gene, which allowed for wide range of expression modulation of the reporter protein, including the most difficult to achieve dynamic switch to an OFF state. Out work provides a frame for achieving independent modulation of gene expression, by only including an independent control circuit expressing synthetic sRNAs, without altering the structure of existing genetic circuits.

show abstract

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Cited by 10 publications

References 34 publications

Orthogonal Regulatory Circuits for Escherichia coli Based on the γ-Butyrolactone System of Streptomyces coelicolor

Orthogonal Regulatory Circuits for Escherichia coli Based on the γ-Butyrolactone System of Streptomyces coelicolor

Gene Expression Knockdown by Modulating Synthetic Small RNA Expression in Escherichia coli

Tailored synthetic sRNAs dynamically tune multilayer genetic circuits

Contact Info

Product

Resources

About