Advertisement

Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli

  • Rikuto Kamiura
  • Yoshihiro ToyaEmail author
  • Fumio Matsuda
  • Hiroshi ShimizuEmail author
Original Research Paper
  • 86 Downloads

Abstract

Objectives

Fine-tuning of enzyme expression at low levels is an important challenge for metabolic engineers. Here, theophylline-inducible riboswitch for translational regulation was evaluated. The background expression, translation rate, and time delay for its induction was reported.

Results

To evaluate the effect of the amount of mRNA on its translation rate, transcription of the riboswitch RNA with red fluorescent protein (RFP) was controlled by the lac system with addition of isopropyl β-d-1-thiogalactopyranoside in Escherichia coli. Regardless of the amount of riboswitch mRNA, the translation of RFP was completely suppressed without theophylline during both growth and stationary phases. Furthermore, a strong positive correlation between theophylline concentration (0 to 1 mM) and specific RFP production rate was observed. The specific RFP production rate with the riboswitch was approximately 2.3% of that without the riboswitch. Furthermore, 60 min of time delay for RFP expression was observed after adding theophylline during the stationary phase.

Conclusion

Theophylline-inducible riboswitch precisely controls protein translation at low expression levels with significantly low background expression. It can emerge as a powerful tool for fine tuning of enzyme expression.

Keywords

Riboswitch Theophylline Escherichia coli Lac system Growth phase Stationary phase 

Notes

Acknowledgements

This work was supported by Grant-in-Aid for Young Scientists (B) No. 16K18298; a Japan Science and Technology Agency (JST)-Mirai Program Grant Number JPMJMI17EJ, Japan.

Supporting information

Fig. S1—The sequence of theophylline inducible riboswitch and RFP gene. The underlined sequence represents theophylline-inducible riboswitch sequence used in this study. The following sequence represents RFP gene.

Fig. S2—Specific growth rate (μ) of IRSrfp strain under various conditions of theophylline and IPTG. With/without induction of theophylline-inducible riboswitch, specific growth rate of the IRSrfp strain was calculated from 3 to 6 h as an indicator of the growth. These were calculated in the same experiment as Fig. 4 (calculation of specific RFP product rate). Among all condition of theophylline (riboswitch induction) and IPTG (lacI induction), there was no difference of the growth. Error bars represent SD (n = 3).

Supplementary material

10529_2019_2672_MOESM1_ESM.pdf (323 kb)
Supplementary material 1 (PDF 323 kb)

References

  1. Anthony LC, Suzuki H, Filutowicz M (2004) Tightly regulated vectors for the cloning and expression of toxic genes. J Microbiol Methods 58:243–250.  https://doi.org/10.1016/j.mimet.2004.04.003 CrossRefGoogle Scholar
  2. Chao YP, Liao JC (1993) Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli. Appl Environ Microbiol 59:4261–4265Google Scholar
  3. Chubukov V, Desmarais JJ, Wang G, Chan LJG, Baidoo EE, Petzold CJ, Keasling JD, Mukhopadhyay A (2017) Engineering glucose metabolism of Escherichia coli under nitrogen starvation. NPJ Syst Biol Appl 3:16035.  https://doi.org/10.1038/npjsba.2016.35 CrossRefGoogle Scholar
  4. Desai SH, Rabinovitch-Deere CA, Tashiro Y, Atsumi S (2014) Isobutanol production from cellobiose in Escherichia coli. Appl Microbiol Biothehcnol 98:3727–3736.  https://doi.org/10.1007/s00253-013-5504-7 CrossRefGoogle Scholar
  5. Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59.  https://doi.org/10.1016/0022-2836(91)90856-2 CrossRefGoogle Scholar
  6. Farmer WR, Liao JC (2000) Improving lycopene production in Escherichia coli by engineering metabolic control. Nat Biotechnol 18:533–537.  https://doi.org/10.1038/75398 CrossRefGoogle Scholar
  7. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD (2011) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12.  https://doi.org/10.1186/1754-1611-5-12 CrossRefGoogle Scholar
  8. Llanes B, McFall E (1969) Role of lac genes in induction of beta-galactosidase synthesis by galactose. J Bacteriol 97:223–229Google Scholar
  9. Long CP, Gonzalez JE, Feist AM, Palsson BO, Antoniewicz MR (2018) Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli. Proc Natl Acad Sci USA 115:222–227.  https://doi.org/10.1073/pnas.1716056115 CrossRefGoogle Scholar
  10. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298.  https://doi.org/10.1006/jmbi.1996.0399 CrossRefGoogle Scholar
  11. Ohbayashi R, Akai H, Yoshikawa H, Hess WR, Watanabe S (2016) A tightly inducible riboswitch system in Synechocystis sp. PCC 6803. J Gen Appl Microbiol 62:154–159.  https://doi.org/10.2323/jgam.2016.02.002 CrossRefGoogle Scholar
  12. Okahashi N, Matsuda F, Yoshikawa K, Shirai T, Matsumoto Y, Wada M, Shimizu H (2017) Metabolic engineering of isopropyl alcohol-producing Escherichia coli strains with 13C-metabolic flux analysis. Biotechnol Bioeng 114:2782–2793.  https://doi.org/10.1002/bit.26390 CrossRefGoogle Scholar
  13. Orth P, Schnappinger D, Hillen W, Saenger W, Hinrichs W (2000) Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat Struct Biol 7:215–219.  https://doi.org/10.1038/73324 CrossRefGoogle Scholar
  14. Suess B, Fink B, Berens C, Stentz R, Hillen W (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 32:1610–1614CrossRefGoogle Scholar
  15. Tokuyama K, Ohno S, Yoshikawa K, Hirasawa T, Tanaka S, Furusawa C, Shimizu H (2014) Increased 3-hydroxypropionic acid production from glycerol, by modification of central metabolism in Escherichia coli. Microb Cell Fact 13:64.  https://doi.org/10.1093/nar/gkh321 CrossRefGoogle Scholar
  16. Usui Y, Hirasawa T, Furusawa C, Shirai T, Yamamoto N, Mori H, Shimizu H (2012) Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using (13)C metabolic flux analysis. Microb Cell Fact 11:87.  https://doi.org/10.1186/1475-2859-11-87 CrossRefGoogle Scholar
  17. Wada K, Toya Y, Banno S, Yoshikawa K, Matsuda F, Shimizu H (2016) 13C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli. J Biosci Bioeng 123:177–182.  https://doi.org/10.1016/j.jbiosc.2016.08.001 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Bioinformatic Engineering, Graduate School of Information Science and TechnologyOsaka UniversitySuitaJapan

Personalised recommendations