Construction of a pathway-independent three-module self-induced regulatory system and its application in...

Dynamic regulatory systems are promising tools for the biosynthesis of high-value chemicals. However, the construction of pathway-independent multimodule self-induced regulatory systems capable of modulating complex metabolic pathways remains challenging. In this study, we developed a multimodule self-induced regulatory system based on two orthogonal quorum sensing (QS) and a stationary-phase sensing system. We optimized the Las and Tra* QS response times by tuning acyl-homoserine lactone (AHL) synthase expression, identified a stationary-phase sensor with a higher output, and integrated these into a pathway-independent multimodule self-induced regulatory system. This system successfully enabled the sequential expression of multiple genes at specified time intervals under autoinduction conditions. Ultimately, this system, combined with a small RNA inhibition module, was employed to produce 3-hydroxypropionic acid (3-HP), which sequentially regulated the three modules of the tricarboxylic acid cycle, fatty acid metabolism, and 3-HP synthesis. Application of this system resulted in a 3-HP titer of 7.0 g/L in shake flask fermentation, which was 27% higher than that of the base strain using a static regulatory strategy, while also improving bacterial growth. This application validates the potential of the developed multimodule self-induced regulatory system to regulate multigene expression in complex microbial metabolic networks.