Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown

Yang, Dongsoo; Yoo, Seung Min; Gu, Changdai; Ryu, Jae Yong; Lee, Jae Eun; Lee, Sang Yup

doi:10.1016/j.ymben.2019.04.003

Cited by 40 publications

(16 citation statements)

References 39 publications

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“…When it comes to regulating gene expression, sRNA stands up as an efficient and easy-to-engineer tool. It has been intensely used to engineer bacteria for gene knockdown by preventing translation initiation (Hoynes-O'Connor and Moon, 2016;Liu et al, 2014;Na et al, 2013;Noh et al, 2017;Yang et al, 2019). Activation of gene expression has been achieved by engineering two RNA molecules, the sRNA and a toehold switch (for translation initiation control) or a Sense target RNA (for transcription termination control) (Chappell et al, 2015a;Green et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is Na et al, 2013;Noh et al, 2017;Sun et al, 2019;Yang et al, 2019Yang et al, , 2015. In order to demonstrate the potential of the riboswitch-targeting sRNA for metabolic engineering, we tested the sRNA ability to improve the riboflavin production directly by targeting the rib operon, and indirectly by targeting the purine supply (Figure 4).…”

Section: Engineering An Improved Riboflavin Producer Strainmentioning

confidence: 99%

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Targeting riboswitches with synthetic small RNAs for metabolic engineering

Lins

Amorim

Corrêa

et al. 2021

Preprint

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Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA (sRNA) tool to target bacterial riboswitches and activate gene expression. The synthetic sRNA is suitable for the regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used sRNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic sRNA to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the riboswitch-targeting sRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

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Section: Discussionmentioning

confidence: 99%

Section: Engineering An Improved Riboflavin Producer Strainmentioning

confidence: 99%

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Lins

Amorim

Corrêa

et al. 2021

Preprint

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“…The recombinant E. coli strain based on FBA produced a 6-fold increase in violacein yield. The synthetic small regulatory RNA (sRNA) technology has been successfully used for genome-scale screening of beneficial knockdown gene targets in a violacein producing E. coli strain (Yang et al, 2019). As a result, knocking down ytfR (encoding sugar ABC transporter) increased 600% violacein titer.…”

Section: Tryptophan Derivativesmentioning

confidence: 99%

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

Shen

Niu

Yan

et al. 2020

Front. Bioeng. Biotechnol.

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Aromatic compounds derived from aromatic amino acids are an important class of diverse chemicals with a wide range of industrial and commercial applications. They are currently produced via petrochemical processes, which are not sustainable and eco-friendly. In the past decades, significant progress has been made in the construction of microbial cell factories capable of effectively converting renewable carbon sources into value-added aromatics. Here, we systematically and comprehensively review the recent advancements in metabolic engineering and synthetic biology in the microbial production of aromatic amino acid derivatives, stilbenes, and benzylisoquinoline alkaloids. The future outlook concerning the engineering of microbial cell factories for the production of aromatic compounds is also discussed.

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“…31−42 A recently developed expanded synthetic sRNA expression platforms allowed rapid, multiplexed, and genome-scale target gene knockdown in engineered Escherichia coli without concerning plasmid compatibility. 43 Also, effective knockdown of essential genes is possible by employing the sRNA technology, which is not easy using the conventional knockout techniques. CRISPR interference (CRISPRi) can also knockdown target gene expression, 44 but is not as efficient as the sRNA technology.…”

mentioning

confidence: 99%

“…The Hfq-binding region enhances the ability of sRNAs to bind target mRNAs, and facilitates mRNA degradation by recruiting RNase E. − The Rho-independent transcription terminator is a GC-rich palindromic sequence followed by a stretch of U nucleotides that terminates the synthesis of sRNAs to produce discrete sRNA molecules. Much effort has been exerted on developing a method to modulate gene repression by modifying natural sRNA scaffold. ,,− Notably, the recent development of synthetic sRNAs has paved the way for more practical and wider use of sRNA in metabolic engineering and synthetic biology that allows in trans high-throughput gene expression control without any chromosomal modification. − A recently developed expanded synthetic sRNA expression platforms allowed rapid, multiplexed, and genome-scale target gene knockdown in engineered Escherichia coli without concerning plasmid compatibility . Also, effective knockdown of essential genes is possible by employing the sRNA technology, which is not easy using the conventional knockout techniques.…”

mentioning

confidence: 99%

Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs

et al. 2019

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Gene expression regulation in broad-spectrum range is critical for constructing cell factories and genetic circuits to balance and control system-wide fluxes. Synthetic small regulatory RNAs (sRNAs) effectively regulate gene expression at the translational level by modulating an mRNA-binding chance and sRNA abundance; however, it can control target gene expression only within the limit of the intrinsic repression ability of sRNAs. Here, we systematically mutated a SgrS scaffold as a model sRNA by dividing the Hfq-binding module of the sRNA into the three regions: the A/U-rich sequence, the stem, and the hairpin loop, and examined how efficiently the mutants suppressed DsRed2 expression. By doing this, we found that a scaffold with an altered A/U-rich sequence (CUUU) and stem length and that with altered A/U-rich sequence (GCAC) showed a 3-fold stronger and a 3-fold weaker repression than the original scaffold, respectively. For practical application of altered scaffolds, proof-of-concept experiments were performed by constructing a library of 67 synthetic sRNAs with the strongest scaffold, each one targeting a different rationally selected gene, and using this library to enhance cadaverine production in Escherichia coli, yielding in 27% increase (1.67 g/L in flask cultivation, 13.7 g/L in fed-batch cultivation). Synthetic sRNAs with engineered sRNA scaffolds could be useful in modulating gene expression for strain improvement.

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Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown

Cited by 40 publications

References 39 publications

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs

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