Controllable gene expression systems that are orthogonal to the host’s native gene regulation network are invaluable tools for synthetic biology. In Ralstonia eutropha H16, such systems are extremely limited despite the importance of this organism in microbiological research and biotechnological application. Here we developed an anhydrotetracycline (aTc)-inducible gene expression system, which is composed of a synthetic promoter containing the operator tetO, the repressor TetR, and the inducer aTc. Using a reporter-activity based promoter library screen, we first identified the active hybrids between the tetO operators and the R. eutropha native rrsC promoter (P_rrsC). Next, we showed that the hybrid promoters are repressable by TetR. To optimize the dynamic range of the system, a high-throughput screening of 300 mutants of R. eutropha phaC1 promoter was conducted to identify suitable promoters to tune the tetR expression level. The final controllable expression system contains the modified P_rrsC with two copies of the tetO1 operator integrated and the tetR driven by the mutated P_phaC1. The system has decreased basal expression level and can be tuned by different aTc concentrations with greater than 10-fold dynamic range. The system was used to alleviate cellular toxicity caused by AlsS overexpression, which impeded our metabolic engineering work on isobutanol and 3-methyl-1-butanol production in R. eutropha H16.

中文翻译：

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富营养小球藻H16的合成脱水四环素可控基因表达系统

与宿主的天然基因调控网络正交的可控基因表达系统是合成生物学的宝贵工具。尽管该生物在微生物学研究和生物技术应用中很重要，但在富营养的Ralstonia H16中，此类系统极为有限。在这里，我们开发了脱水四环素（aTc）诱导型基因表达系统，该系统由包含操纵子tetO，阻遏物TetR和诱导子aTc的合成启动子组成。利用报告活动基于启动库屏幕上，我们首先确定之间的活跃杂交tetO的运营商和富氧罗尔斯本地rrsC启动子（P _rrsC）。接下来，我们表明杂合启动子可被TetR抑制。为了优化系统的动态范围，进行了3​​00个富营养富营养芽孢杆菌phaC1启动子突变体的高通量筛选，以鉴定合适的启动子来调节tetR表达水平。最终的可控表达系统包含修饰的P _rrsC，其中整合了tetO1算子的两个副本以及由突变的P _phaC1驱动的tetR。该系统具有降低的基础表达水平，并且可以通过具有大于10倍动态范围的不同aTc浓度进行调节。该系统用于减轻由AlsS过表达引起的细胞毒性，这阻碍了我们在富营养R. eutropha H16中生产异丁醇和3-甲基-1-丁醇的代谢工程工作。