State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, Jinan 250100, China.
CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
Molecules. 2022 Jan 18;27(3):617. doi: 10.3390/molecules27030617.
Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene () and PHB synthesis gene () expression. Consequently, the strain RBS34, which has high expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.
已经开发了几种策略，包括诱导剂添加和生物传感器使用，用于动态调节。然而，现有策略的毒性、成本和灵活性要求开发更好的技术。在本研究中，我们设计了一个光遗传学双开关系统，并将其应用于提高聚羟基丁酸酯（PHB）的产量。首先，选择了一个优化的光色适应传感器/调节剂（RBS10-CcaS#10-CcaR）系统（包含优化的核糖体结合位点（RBS）、光感觉蛋白 CcaS 和反应调节剂 CcaR），用于在绿光激活和红光抑制之间大约 10 倍的宽感应范围。RBS10-CcaS#10-CcaR 系统与蓝光照激活的 YF1-FixJ-PhlF 系统（包含组氨酸激酶 YF1、反应调节剂 FixJ 和抑制剂 PhlF）相结合，该系统经过工程设计以减少串扰。最后，光遗传学双开关系统用于通过调节柠檬酸合酶基因（）和 PHB 合成基因（）表达的序列和间隔来重新布线 PHB 生产的代谢通量。结果，具有高表达和 3 小时时间滞后的菌株 RBS34 实现了最高 16.6wt%的 PHB 含量，约为 F34（在 0 小时表达）的 3 倍。结果表明，光遗传学双开关系统被验证为提高 PHB 产量的实用且方便的工具。
