Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces

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 similar to 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.