ObjectivesFine-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.ResultsTo 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 -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 1mM) 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, 60min of time delay for RFP expression was observed after adding theophylline during the stationary phase.ConclusionTheophylline-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.