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

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Abstract

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ PR promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the PR promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type PR promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.

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Authors and Affiliations

  1. School of Integrative Engineering, Chung-Ang University, Seoul, 06974, Korea

    Minhui Sung, Ren Jun & Dokyun Na

  2. Department of Chemical and Biomolecular Engineering (BK21 plus Program), KAIST, Daejeon, 34141, Korea

    Seung Min Yoo, Jae Eun Lee & Sang Yup Lee

Authors
  1. Minhui Sung
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  2. Seung Min Yoo
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  3. Ren Jun
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  4. Jae Eun Lee
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  5. Sang Yup Lee
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  6. Dokyun Na
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Correspondence to Sang Yup Lee or Dokyun Na.

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These authors equally contributed to this work.

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Sung, M., Yoo, S.M., Jun, R. et al. Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering. Biotechnol Bioproc E 21, 483–490 (2016). https://doi.org/10.1007/s12257-016-0245-y

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  • DOI: https://doi.org/10.1007/s12257-016-0245-y

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