Zhang Jie, Sonnenschein Nikolaus, Pihl Thomas P B, Pedersen Kasper R, Jensen Michael K, Keasling Jay D
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
Joint BioEnergy Institute , Emeryville, California 94608, United States.
ACS Synth Biol. 2016 Dec 16;5(12):1546-1556. doi: 10.1021/acssynbio.6b00135. Epub 2016 Jul 25.
Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP redox biosensor in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP ratios from mixed cell populations. We show that the combination of exploitation and rational engineering of native signaling components is applicable for diagnosis, regulation, and selection of cellular redox states.
基因编码生物传感器已成为及时、精确地在体内评估细胞代谢的强大工具。特别是,能够将细胞间信号与下游信号反应联系起来的生物传感器目前在健康科学和生物技术领域备受关注。然而,仍需要对更多生物传感器进行生物勘探和工程设计,以实现对特定细胞状态的实时监测和对下游激活的控制。在本研究中,我们报告了一种基于转录因子的NADPH/NADP氧化还原生物传感器在酿酒酵母中的工程设计与应用。利用该生物传感器,我们能够通过化学诱导监测氧化应激的原因,以及基因操作引起的NADPH/NADP比率变化。由于该生物传感器具有调节潜力,我们还表明它可以通过激活NADPH再生来应对NADPH缺乏。最后,我们将生物传感器与剂量敏感基因(DSG)的表达相结合,从而创建了一种新型的可调谐传感器选择器,可用于从混合细胞群体中合成选择具有更高NADPH/NADP比率的细胞。我们表明,对天然信号成分的开发与合理工程设计相结合,适用于细胞氧化还原状态的诊断、调节和选择。