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Synthetic fused sRNA for the simultaneous repression of multiple genes

  • Applied Genetics and Molecular Biotechnology
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Abstract

Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid–producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.

Key points

• Synthetic fusion sRNA (fsRNA)–based system was constructed for the repression of multiple target genes.

• fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.

• The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).

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Data availability

All datasets obtained for this study are included in the manuscript/supplementary material.

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Funding

This research was supported by an NRF grant funded by the Ministry of Science and ICT (NRF-2022R1A2C2004292) and the Chung-Ang University Research Scholarship Grants in 2021.

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Authors and Affiliations

  1. School of Integrative Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06974, Republic of Korea

    Jinho Yeom, Jong Seong Park, Yong Min Jeon, Beom Seop Song & Seung Min Yoo

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  1. Jinho Yeom
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  2. Jong Seong Park
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  3. Yong Min Jeon
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  4. Beom Seop Song
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  5. Seung Min Yoo
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Contributions

SMY and JY conceived the project and designed the experiments. JY, JSP, YMJ and BSS performed the experiments. SMY, JY and JSP analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Seung Min Yoo.

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Yeom, J., Park, J.S., Jeon, Y.M. et al. Synthetic fused sRNA for the simultaneous repression of multiple genes. Appl Microbiol Biotechnol 106, 2517–2527 (2022). https://doi.org/10.1007/s00253-022-11867-5

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  • DOI: https://doi.org/10.1007/s00253-022-11867-5

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