Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, PtrpO1-TrpR1 and PtetO1-TetR. In this genetic inverter, the repression of TetR towards PtetO1 could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of PtrpO1-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.

中文翻译：

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构建基于转录抑制子的遗传反相器，用于大肠杆菌中色氨酸衍生途径的调节


高效的微生物细胞工厂需要复杂而精确的代谢调节来优化生产，这可以通过实施具有多功能功能的调节遗传电路得到显着帮助。然而，在宿主菌株中构建功能多样的遗传电路具有挑战性。特别是，由于难以逆转转录阻遏物的抑制特性，因此很少探索基于转录阻遏物的功能多样化。为了解决这个问题，我们提出了一种设计逻辑来创建基于转录抑制子的遗传反相器以实现功能富集。作为概念证明，通过整合两组转录抑制子 PtrpO1-TrpR1 和 PtetO1-TetR 构建了色氨酸诱导的遗传反相器。在这个遗传反相器中，在色氨酸存在的情况下，色氨酸-TrpR1复合物可以减轻TetR对PtetO1的抑制，从而导致激活的输出。随后，我们优化了逆变器的动态性能，构建了色氨酸触发的动态激活系统。 PtrpO1-TrpR1 的原始抑制功能与反相器变体的进一步耦合实现了色氨酸触发的双功能调节系统。最后，动态调节系统实现了色氨酸生产监测。这些系统还使色氨酸衍生物色胺和紫罗素的效价分别显着提高了 2.0 倍和 7.4 倍。基因反相器的成功设计和应用增强了转录抑制子的适用性。