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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
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
Chao YP, Liao JC (1993) Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli. Appl Environ Microbiol 59:4261–4265
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
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
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
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
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
Llanes B, McFall E (1969) Role of lac genes in induction of beta-galactosidase synthesis by galactose. J Bacteriol 97:223–229
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
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
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
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
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
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 32:1610–1614
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
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
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
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.
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).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
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). https://doi.org/10.1007/s10529-019-02672-8
- Escherichia coli
- Lac system
- Growth phase
- Stationary phase