Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces
Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces
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开发基于内源群体感应的 CRISPRi 电路，用于链霉菌中多个靶标的自主和可调动态调节
DOI：
10.1093/nar/gkaa602
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发表时间：
2020-08-20
影响因子：
14.9
通讯作者：
Jiang, Weihong
中科院分区：
文献类型：
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作者：
Tian, Jinzhong;Yang, Gaohua;Jiang, Weihong
摘要群体感应（QS）介导的动态调节已成为优化微生物产品效价的有效策略。然而，这些基于QS的电路通常是在异源系统上创建的，并且需要通过繁琐的测试/优化过程进行仔细的调整。这阻碍了它们在工业微生物中的应用。在这里，我们设计了一个新的QS电路，直接整合内源性QS系统与CRISPRi（命名为EQCi）的工业雷帕霉素生产菌株雷帕霉素链霉菌。EQCi结合了QS系统和CRISPRi的优势，能够同时对多个目标进行可调、自主和动态调节。使用EQCi，我们分别下调三个关键节点的基本途径，转向代谢通量对雷帕霉素的生物合成，并显着增加其滴度。进一步应用EQCi以同时调节这三个具有微调阻遏强度的关键节点，将雷帕霉素滴度提高了1.660%，达到了最高的报道滴度（1836 ± 191 mg/l）。值得注意的是，与导致生长停滞和次优雷帕霉素滴度的静态工程化策略相比，基于EQCi的调节显著促进雷帕霉素滴度而不影响细胞生长，表明它可以实现必需途径和产物合成之间的权衡。总之，本研究为菌种改良提供了一种方便有效的策略，并显示出在其他工业微生物中的应用潜力。
Abstract Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms.