Targeting riboswitches with synthetic small RNAs for metabolic engineering

Lins, Milca Rachel da Costa Ribeiro; Amorim, Laura Araujo da Silva; Corrêa, Graciely Gomes; Picão, Bruno Willian; Mack, Matthias; Cerri, Marcel O.; Pedrolli, Danielle Biscaro

doi:10.1016/j.ymben.2021.09.003

Cited by 7 publications

(4 citation statements)

References 41 publications

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“…This work achieved artificial sRNA-mediated transcription activation in vivo first time [ 9 ]. Lins et al firstly implemented STARs in B. subtilis , called riboswitch-targeting sRNAs (rtRNA) [ 111 ]. By targeting at terminator stem-loop, rtRNAs activate gene expression by turning riboswitches into ON state ( Fig.…”

Section: Artificial Srna Design and Applicationmentioning

confidence: 99%

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Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

Peng,

Yin,

Zuo

et al. 2024

Synthetic and Systems Biotechnology

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Section: Artificial Srna Design and Applicationmentioning

confidence: 99%

“…6 A). rtRNAs could both work in vitro and in vivo , increasing gene expression up to 103-fold [ 111 ]. This work effectively engineered a natural RNA transcriptional repressor as well as the ability to convert intrinsic terminators into transcription-on regulators.…”

Section: Artificial Srna Design and Applicationmentioning

confidence: 99%

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

Peng,

Yin,

Zuo

et al. 2024

Synthetic and Systems Biotechnology

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“…Understanding the sRNA-mediated regulation in bacteria has provided opportunities for applying synthetic biology and metabolic applications as a tool for fine-tuning gene expression with riboswitches ( 12 , 13 ) and a synthetic sRNA scaffold ( 14 , 15 ), manipulating stress-responsive regulation ( 16 ), developing synthetic biosensors to detect specific molecules or environmental changes ( 17 ) and altering metabolic pathways for production of the desired chemical by overexpressing sRNAs ( 18 , 19 ). However, those sRNAs in the biotechnological applications have been used to act as synthetic regulators to modulate downstream regulatory cascades, rather than as direct controlling targets from the upstream regulation.…”

Section: Introductionmentioning

confidence: 99%

CRISPR–dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria

Ko,

Woo

2023

Nucleic Acids Research

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Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR–dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications.

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“…Different strategies, including metabolic engineering (Koch et al, 2019), synthetic biology (Liu et al, 2019), enzyme engineering (R. Weiss et al, 2020), material science (Tang et al, 2020), and bioinformatics (Dixon et al, 2021), have been developed to increase the efficiency of microbial cell factories (Zhou et al, 2015). Among these, metabolic engineering at the DNA level (Wang, 2012), RNA level (Lins et al, 2021), protein level (Stein & Alexandrov, 2015), metabolite level (Seok et al, 2021), and cell level (Stephens & Bentley, 2020) represent the most direct approaches to enhance microbial cell factory efficiency. At the cell level, microbial consortia can affect factory efficiency through the regulation of metabolic division (Zhou et al, 2015), population quality control (Hartline et al, 2020), consortium structure (Liao et al, 2019), and altruism interaction (Honjo et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Engineering Escherichia coli asymmetry distribution‐based synthetic consortium for shikimate production

Ding

Guo

et al. 2022

Biotech & Bioengineering

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Microbial consortia constitute a promising tool for achieving high-value chemical bio-production. However, customizing the consortium ratio remains challenging.Herein, an asymmetry distribution-based synthetic consortium (ADSC) was developed to switch cell phenotypes using shikimate synthesis for proof of concept. First, the cell pole-organizing protein PopZ was screened as a mediator of asymmetric protein distribution in Escherichia coli. The ADSC was then constructed to incorporate PopZmediated asymmetry distribution and a TetR-based transcription repression switch to achieve the dynamical control of microbial population ratio. Finally, the ADSC was used to decouple cell growth from shikimate synthesis by effectively coordinating the ratio of growing cells and production cells at the consortium level, thereby increasing shikimate titer to 30.1 g/L in the 7.5-L bioreactor with a minimal medium. This titer was further improved to 82.5 g/L when using rich medium fermentation. Our results illustrate a novel approach to control consortium structure through ADSC-mediated regulation, highlighting its potential as an efficient strategy for controlling metabolic state in microbes.

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Targeting riboswitches with synthetic small RNAs for metabolic engineering

Cited by 7 publications

References 41 publications

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

CRISPR–dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria

Engineering Escherichia coli asymmetry distribution‐based synthetic consortium for shikimate production

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