Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Biotechnol Lett. 2019 Jul;41(6-7):743-751. doi: 10.1007/s10529-019-02672-8. Epub 2019 Apr 5.
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.
在低水平下精确调控酶表达是代谢工程师面临的一项重要挑战。本研究评估了茶碱诱导型核糖体开关的翻译调控作用。报告了其背景表达、翻译速率和诱导的时滞。
为了评估 mRNA 数量对其翻译速率的影响，在大肠杆菌中通过乳糖系统控制核糖体开关 RNA 的转录，并用异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导。无论核糖体开关 mRNA 的数量多少，在生长和静止期都没有茶碱时，RFP 的翻译完全受到抑制。此外，观察到茶碱浓度（0 至 1 mM）与特定 RFP 生产速率之间存在强烈的正相关关系。带有核糖体开关的 RFP 特异性产量约为没有核糖体开关时的 2.3%。此外，在静止期添加茶碱后，RFP 表达有 60 分钟的时滞。
茶碱诱导型核糖体开关在低表达水平下可精确调控蛋白翻译，且背景表达水平极低。它可能成为精细调控酶表达的有力工具。
