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Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs

Nature Biotechnology volume 31, pages 170–174 (2013)Cite this article

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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.

References

  1. Qian, Z.-G., Xia, X.-X. & Lee, S.Y. Metabolic engineering of Escherichia coli for the production of cadaverine: a five-carbon diamine. Biotechnol. Bioeng. 108, 93–103 (2011).

    Article  CAS  Google Scholar 

  2. Pfleger, B.F., Pitera, D.J., Smolke, C.D. & Keasling, J.D. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027–1032 (2006).

    Article  CAS  Google Scholar 

  3. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  CAS  Google Scholar 

  4. Sharon, E. et al. Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nat. Biotechnol. 30, 521–530 (2012).

    Article  CAS  Google Scholar 

  5. Sandoval, N.R. et al. Strategy for directing combinatorial genome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 109, 10540–10545 (2012).

    Article  CAS  Google Scholar 

  6. Ho, C.H. et al. A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds. Nat. Biotechnol. 27, 369–377 (2009).

    Article  CAS  Google Scholar 

  7. Kress, W.J. & Erickson, D.L. DNA barcodes: genes, genomics, and bioinformatics. Proc. Natl. Acad. Sci. USA 105, 2761–2762 (2008).

    Article  CAS  Google Scholar 

  8. Saito, H. & Inoue, T. Synthetic biology with RNA motifs. Int. J. Biochem. Cell Biol. 41, 398–404 (2009).

    Article  CAS  Google Scholar 

  9. Desai, S.K. & Gallivan, J.P. Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J. Am. Chem. Soc. 126, 13247–13254 (2004).

    Article  CAS  Google Scholar 

  10. Isaacs, F.J. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotechnol. 22, 841–847 (2004).

    Article  CAS  Google Scholar 

  11. Carothers, J.M., Goler, J.A., Juminaga, D. & Keasling, J.D. Model-driven engineering of RNA devices to quantitatively program gene expression. Science 334, 1716–1719 (2011).

    Article  CAS  Google Scholar 

  12. Sharma, V., Yamamura, A. & Yokobayashi, Y. Engineering artificial small RNAs for conditional gene silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13 (2012).

    Article  CAS  Google Scholar 

  13. Liang, J.C., Bloom, R.J. & Smolke, C.D. Engineering biological systems with synthetic RNA molecules. Mol. Cell 43, 915–926 (2011).

    Article  CAS  Google Scholar 

  14. Win, M.N. & Smolke, C.D. Higher-order cellular information processing with synthetic RNA devices. Science 322, 456–460 (2008).

    Article  CAS  Google Scholar 

  15. Lucks, J.B., Qi, L., Mutalik, V.K., Wang, D. & Arkin, A.P. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc. Natl. Acad. Sci. USA 108, 8617–8622 (2011).

    Article  CAS  Google Scholar 

  16. Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A. Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470–474 (2011).

    Article  CAS  Google Scholar 

  17. Kang, Z., Wang, X., Li, Y., Wang, Q. & Qi, Q. Small RNA RyhB as a potential tool used for metabolic engineering in Escherichia coli. Biotechnol. Lett. 34, 527–531 (2012).

    Article  CAS  Google Scholar 

  18. Gottesman, S. The small RNA regulators of Escherichia coli: Roles and mechanisms. Annu. Rev. Microbiol. 58, 303–328 (2004).

    Article  CAS  Google Scholar 

  19. Man, S. et al. Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria. Nucleic Acids Res. 39, e50 (2011).

    Article  CAS  Google Scholar 

  20. Urban, J.H. & Vogel, J. Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res. 35, 1018–1037 (2007).

    Article  CAS  Google Scholar 

  21. Aiba, H. Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. Opin. Microbiol. 10, 134–139 (2007).

    Article  CAS  Google Scholar 

  22. Pichon, C., du Merle, L., Caliot, M.E., Trieu-Cuot, P. & Le Bouguénec, C. An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains. Nucleic Acids Res. 40, 2846–2861 (2012).

    Article  CAS  Google Scholar 

  23. Culver, G.M. Meanderings of the mRNA through the ribosome. Structure 9, 751–758 (2001).

    Article  CAS  Google Scholar 

  24. Na, D., Lee, S. & Lee, D. Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with a desired expression level in prokaryotes. BMC Syst. Biol. 4, 71 (2010).

    Article  Google Scholar 

  25. Lütke-Eversloh, T. & Stephanopoulos, G. L-tyrosine production by deregulated strains of Escherichia coli. Appl. Microbiol. Biotechnol. 75, 103–110 (2007).

    Article  Google Scholar 

  26. Juminaga, D. et al. Modular engineering of L-tyrosine production in Escherichia coli. Appl. Environ. Microbiol. 78, 89–98 (2012).

    Article  CAS  Google Scholar 

  27. Lee, S.Y., Lee, K.M., Chan, H.N. & Steinbüchel, A. Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes. Biotechnol. Bioeng. 44, 1337–1347 (1994).

    Article  CAS  Google Scholar 

  28. Lütke-Eversloh, T. & Stephanopoulos, G. Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression. Metab. Eng. 10, 69–77 (2008).

    Article  Google Scholar 

  29. Hua, Q., Yang, C., Baba, T., Mori, H. & Shimizu, K. Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J. Bacteriol. 185, 7053–7067 (2003).

    Article  CAS  Google Scholar 

  30. Kadir, T.A., Mannan, A., Kierzek, A., McFadden, J. & Shimizu, K. Modeling and simulation of the main metabolism in Escherichia coli and its several single-gene knockout mutants with experimental verification. Microb. Cell Fact. 9, 88 (2010).

    Article  Google Scholar 

  31. Markham, N.R. & Zuker, M. UNAFold: software for nucleic acid folding and hybridization. Methods Mol. Biol. 453, 3–31 (2008).

    Article  CAS  Google Scholar 

  32. Jana, N.K., Roy, S., Bhattacharyya, B. & Mandal, N.C. Amino acid changes in the repressor of bacteriophage λ due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor. Protein Eng. 12, 225–233 (1999).

    Article  CAS  Google Scholar 

  33. Xia, X.-X. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. USA 107, 14059–14063 (2010).

    Article  CAS  Google Scholar 

  34. Pasquali, C. et al. Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database. Electrophoresis 17, 547–555 (1996).

    Article  CAS  Google Scholar 

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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 and Affiliations

  1. Department of Chemical and Biomolecular Engineering (BK21 program), Metabolic and Biomolecular Engineering National Research Laboratory, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Dokyun Na, Seung Min Yoo, Hannah Chung, Hyegwon Park, Jin Hwan Park & Sang Yup Lee

  2. BioProcess Engineering Research Center, Bioinformatics Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Dokyun Na, Seung Min Yoo, Jin Hwan Park & Sang Yup Lee

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  1. Dokyun Na
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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.

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Correspondence to Sang Yup Lee.

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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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