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Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs

Abstract

Small regulatory RNAs (sRNAs) regulate gene expression in bacteria. We designed synthetic sRNAs to identify and modulate the expression of target genes for metabolic engineering in Escherichia coli. Using synthetic sRNAs for the combinatorial knockdown of four candidate genes in 14 different strains, we isolated an engineered E. coli strain (tyrR- and csrA-repressed S17-1) capable of producing 2 g per liter of tyrosine. Using a library of 130 synthetic sRNAs, we also identified chromosomal gene targets that enabled substantial increases in cadaverine production. Repression of murE led to a 55% increase in cadaverine production compared to the reported engineered strain (XQ56 harboring the plasmid p15CadA)1. The design principles and the engineering strategy using synthetic sRNAs reported here are generalizable to other bacteria and applicable in developing superior producer strains. The ability to fine-tune target genes with designed sRNAs provides substantial advantages over gene-knockout strategies and other large-scale target identification strategies owing to its easy implementation, ability to modulate chromosomal gene expression without modifying those genes and because it does not require construction of strain libraries.

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Figure 1: Design principles for synthetic sRNAs.
Figure 2: Metabolic engineering of E. coli for the production of tyrosine using a synthetic sRNA strategy.
Figure 3: Synthetic sRNA–based strategy for large-scale target identification and fine-tuning of gene expression for enhanced cadaverine production.

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Acknowledgements

We would like to thank M.-H. Lee for measuring tyrosine and cadaverine concentrations using high-performance liquid chromatography, Y.H. Lee for 2D-PAGE experiments, J.A. Im for large-scale cloning of synthetic sRNAs and cultivation experiments, and S.J. Choi for fermentation experiments. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea; and the World Class University program (R32-2008-000-10142-0) of MEST.

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Authors

Contributions

S.Y.L. and D.N. conceived of the project. D.N. designed the structure of synthetic sRNAs and performed sRNA construction and evaluation and metabolic engineering experiments. S.M.Y. performed sRNA construction and evaluation and metabolic engineering experiments. H.C. and H.P. carried out large-scale screening for cadaverine production. J.H.P. constructed the S17-1 knockout strain and performed fermentation experiments. S.Y.L. supervised the project. All authors contributed to the preparation of the manuscript.

Corresponding author

Correspondence to Sang Yup Lee.

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The authors declare no competing financial interests.

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Supplementary Figures 1–19, Supplementary Tables 1–3 and Supplementary Sequences (PDF 4437 kb)

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Na, D., Yoo, S., Chung, H. et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 31, 170–174 (2013). https://doi.org/10.1038/nbt.2461

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