Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression

Abstract

Cyanobacteria are attractive host bacteria for biofuel production because they can covert CO2 to biofuel lipids using only sunlight, water, and inorganic ions. For genetically engineering an ideal cyanobacterium, a synthetic biological approach is promising but few genetic components have been characterized in cyanobacteria. Here for controlling cyanobacterial protein expression, we constructed riboregulators, that one of the post-transcriptional regulators composed of RNAs. Riboregulators harboring a ribosome-binding site suitable for Synechocystis sp. were designed by trial and error using Escherichia coli as host bacteria. The designed riboregulators were effective in Synechocystis sp. as well as E. coli with slight interference on growth only observed in E. coli. They will therefore be useful tools for controlling target gene expression.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3

References

  1. Atsumi S, Higashide W, Liao JC (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 27:1177–1180

    Article  CAS  PubMed  Google Scholar 

  2. Bagdasarian M, Lurz R, Ruckert B, Franklin FC, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247

    Article  CAS  PubMed  Google Scholar 

  3. Callura JM, Dwyer DJ, Isaacs FJ, Cantor CR, Collins JJ (2010) Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proc Natl Acad Sci USA 107:15898–15903

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Callura JM, Cantor CR, Collins JJ (2012) Genetic switchboard for synthetic biology applications. Proc Natl Acad Sci USA 109:5850–5855

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Franch T, Petersen M, Wagner EG, Jacobsen JP, Gerdes K (1999) Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure. J Mol Biol 294:1115–1125

    Article  CAS  PubMed  Google Scholar 

  6. Friedland AE, Lu TK, Wang X, Shi D, Church G, Collins JJ (2009) Synthetic gene networks that count. Science 324:1199–1202

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjo K, Lindblad P (2011) Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods Enzymol 497:539–579

    Article  CAS  PubMed  Google Scholar 

  8. Heidrich N, Brantl S (2003) Antisense-RNA mediated transcriptional attenuation: importance of a U-turn loop structure in the target RNA of plasmid pIP501 for efficient inhibition by the antisense RNA. J Mol Biol 333:917–929

    Article  CAS  PubMed  Google Scholar 

  9. Hjalt TA, Wagner EG (1995) Bulged-out nucleotides protect an antisense RNA from RNase III cleavage. Nucleic Acids Res 23:571–579

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Huang HH, Camsund D, Lindblad P, Heidorn T (2010) Design and characterization of molecular tools for a synthetic biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 38:2577–2593

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Isaacs FJ, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ (2004) Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol 22:841–847

    Article  CAS  PubMed  Google Scholar 

  12. Liu X, Curtiss R III (2009) Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc Natl Acad Sci USA 106:21550–21554

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Liu X, Curtiss R III (2012) Thermorecovery of cyanobacterial fatty acids at elevated temperatures. J Biotechnol 161:445–449

    Article  CAS  PubMed  Google Scholar 

  14. Lopez-Maury L, Garcia-Dominguez M, Florencio FJ, Reyes JC (2002) A two-component signal transduction system involved in nickel sensing in the cyanobacterium Synechocystis sp. PCC 6803. Mol Microbiol 43:247–256

    Article  CAS  PubMed  Google Scholar 

  15. Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res 25:1203–1210

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Mitschke J, Georg J, Scholz I, Sharma CM, Dienst D, Bantscheff J, Voss B, Steglich C, Wilde A, Vogel J et al (2011) An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC 6803. Proc Natl Acad Sci USA 108:2124–2129

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Oliver JW, Machado IM, Yoneda H, Atsumi S (2013) Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proc Natl Acad Sci USA 110:1249–1254

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Rodrigo G, Landrain TE, Jaramillo A (2012) De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells. Proc Natl Acad Sci USA 109:15271–15276

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171–205

    PubMed Central  CAS  PubMed  Google Scholar 

  20. Ying X, Cao Y, Wu J, Liu Q, Cha L, Li W (2011) sTarPicker: a method for efficient prediction of bacterial sRNA targets based on a two-step model for hybridization. PLoS One 6:e22705

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported financially by the Core Research of Evolutional Science & Technology program (CREST) from the Japan Science and Technology Agency (JST).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kazunori Ikebukuro.

Additional information

Koichi Abe and Yuta Sakai.contributed equally to paper.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 15 kb)

Supplementary material 2 (DOCX 11 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Abe, K., Sakai, Y., Nakashima, S. et al. Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression. Biotechnol Lett 36, 287–294 (2014). https://doi.org/10.1007/s10529-013-1352-x

Download citation

Keywords

  • Biofuels
  • Cyanobacteria
  • Post-transcriptional gene regulation
  • Riboregulator
  • Synechocystis
  • Synthetic biology