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



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.


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.


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.

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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).

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Correspondence to Yoshihiro Toya or Hiroshi Shimizu.

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Kamiura, R., Toya, Y., Matsuda, F. et al. Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli. Biotechnol Lett 41, 743–751 (2019).

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  • Riboswitch
  • Theophylline
  • Escherichia coli
  • Lac system
  • Growth phase
  • Stationary phase